Heterodimeric antibodies that bind somatostatin receptor 2

ABSTRACT

The present invention is directed to antibodies, including novel antigen binding domains and heterodimeric antibodies, that bind somatostatin receptor 2 (SSTR2)

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/636,590, filed Jun. 28, 2017 which claims the benefit of U.S.Provisional Application Nos. 62/481,065 filed Apr. 3, 2017, 62/397,322,filed Sep. 20, 2016, 62/355,821, filed Jun. 28, 2016 and 62/355,820,filed Jun. 28, 2016, the contents of which are expressly fullyincorporated by reference in their entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jun. 28, 2017, isnamed 067461-5194-WO_SL.txt and is 2,771,347 bytes in size.

BACKGROUND OF THE INVENTION

Antibody-based therapeutics have been used successfully to treat avariety of diseases, including cancer and autoimmune/inflammatorydisorders. Yet improvements to this class of drugs are still needed,particularly with respect to enhancing their clinical efficacy. Oneavenue being explored is the engineering of additional and novel antigenbinding sites into antibody-based drugs such that a singleimmunoglobulin molecule co-engages two different antigens. Suchnon-native or alternate antibody formats that engage two differentantigens are often referred to as bispecifics. Because the considerablediversity of the antibody variable region (Fv) makes it possible toproduce an Fv that recognizes virtually any molecule, the typicalapproach to bispecific generation is the introduction of new variableregions into the antibody.

A number of alternate antibody formats have been explored for bispecifictargeting (Chames & Baty, 2009, mAbs 1[6]:1-9; Holliger & Hudson, 2005,Nature Biotechnology 23[9]:1126-1136; Kontermann, mAbs 4(2):182 (2012),all of which are expressly incorporated herein by reference). Initially,bispecific antibodies were made by fusing two cell lines that eachproduced a single monoclonal antibody (Milstein et al., 1983, Nature305:537-540). Although the resulting hybrid hybridoma or quadroma didproduce bispecific antibodies, they were only a minor population, andextensive purification was required to isolate the desired antibody. Anengineering solution to this was the use of antibody fragments to makebispecifics. Because such fragments lack the complex quaternarystructure of a full length antibody, variable light and heavy chains canbe linked in single genetic constructs. Antibody fragments of manydifferent forms have been generated, including diabodies, single chaindiabodies, tandem scFv's, and Fab2 bispecifics (Chames & Baty, 2009,mAbs 1[6]:1-9; Holliger & Hudson, 2005, Nature Biotechnology23[9]:1126-1136; expressly incorporated herein by reference). Whilethese formats can be expressed at high levels in bacteria and may havefavorable penetration benefits due to their small size, they clearrapidly in vivo and can present manufacturing obstacles related to theirproduction and stability. A principal cause of these drawbacks is thatantibody fragments typically lack the constant region of the antibodywith its associated functional properties, including larger size, highstability, and binding to various Fc receptors and ligands that maintainlong half-life in serum (i.e. the neonatal Fc receptor FcRn) or serve asbinding sites for purification (i.e. protein A and protein G).

More recent work has attempted to address the shortcomings offragment-based bispecifics by engineering dual binding into full lengthantibody-like formats (Wu et al., 2007, Nature Biotechnology25[11]:1290-1297; U.S. Ser. No. 12/477,711; Michaelson et al., 2009,mAbs 1[2]:128-141; PCT/US2008/074693; Zuo et al., 2000, ProteinEngineering 13[5]:361-367; U.S. Ser. No. 09/865,198; Shen et al., 2006,J Biol Chem 281[16]:10706-10714; Lu et al., 2005, J Biol Chem280[20]:19665-19672; PCT/US2005/025472; expressly incorporated herein byreference). These formats overcome some of the obstacles of the antibodyfragment bispecifics, principally because they contain an Fc region. Onesignificant drawback of these formats is that, because they build newantigen binding sites on top of the homodimeric constant chains, bindingto the new antigen is always bivalent.

For many antigens that are attractive as co-targets in a therapeuticbispecific format, the desired binding is monovalent rather thanbivalent. For many immune receptors, cellular activation is accomplishedby cross-linking of a monovalent binding interaction. The mechanism ofcross-linking is typically mediated by antibody/antigen immunecomplexes, or via effector cell to target cell engagement. For example,the low affinity Fc gamma receptors (FcγRs) such as FcγRIIa, FcγRIIb,and FcγRIIIa bind monovalently to the antibody Fc region. Monovalentbinding does not activate cells expressing these FcγRs; however, uponimmune complexation or cell-to-cell contact, receptors are cross-linkedand clustered on the cell surface, leading to activation. For receptorsresponsible for mediating cellular killing, for example FcγRIIIa onnatural killer (NK) cells, receptor cross-linking and cellularactivation occurs when the effector cell engages the target cell in ahighly avid format (Bowles & Weiner, 2005, J Immunol Methods 304:88-99,expressly incorporated by reference). Similarly, on B cells theinhibitory receptor FcγRIIb downregulates B cell activation only when itengages into an immune complex with the cell surface B-cell receptor(BCR), a mechanism that is mediated by immune complexation of solubleIgG's with the same antigen that is recognized by the BCR (Heyman 2003,Immunol Lett 88[2]:157-161; Smith and Clatworthy, 2010, Nature ReviewsImmunology 10:328-343; expressly incorporated by reference). As anotherexample, CD3 activation of T-cells occurs only when its associatedT-cell receptor (TCR) engages antigen-loaded MHC on antigen presentingcells in a highly avid cell-to-cell synapse (Kuhns et al., 2006,Immunity 24:133-139). Indeed nonspecific bivalent cross-linking of CD3using an anti-CD3 antibody elicits a cytokine storm and toxicity(Perruche et al., 2009, J Immunol 183[2]:953-61; Chatenoud & Bluestone,2007, Nature Reviews Immunology 7:622-632; expressly incorporated byreference). Thus for practical clinical use, the preferred mode of CD3co-engagement for redirected killing of targets cells is monovalentbinding that results in activation only upon engagement with theco-engaged target.

Somatostatins are neuropeptides that act as endogenous inhibitoryregulators. Somatostatins have a broad range of cellular functions suchas inhibition of many secretions, cell proliferation and cell survival(Patel, 1999, Front Neuroendocrinol. 20:157-198). Somatostatins arebroadly distributed in the centeral nervous system, peripheral nervoussystem, pancreas and gut (see, e.g., Watt et al., 2008, Mol CellEndocrinol. 286: 251-261; Epelbaum, 1986, Prog. Neurobiol. 27: 63-100;and Raynor, 1992, Crit. Rev. Neurobiol. 6: 273-289). Somatostatins arealso expressed in neuroendocrine tumors (NETs), such as medullary,thyroid cancer, neuroblastoma, ganglioneuroma, glucagonmas,adenocortical tumors and tumors that appear in the lung, paraganglia,duodenum and some other non-NETs (Volante et al., 2008, Mol. Cell.Endocrinol. 286: 219-229). Somatostatins can elicit effects on targetcells by directly activating somatostatin receptors (SSTRs)(Watt et al.,2008, Mol Cell Endocrinol. 286: 251-261; Pyronnet et al., 2008, Mol.Cell. Endocrinol. 286: 230-237).

Somatostatin receptors (SSTRs) belong to a superfamily of Gprotein-coupled receptors (GPCRs) that each containing a singlepolypeptide chain consisting of extracellular/intracellular domains, andseven transmembrane domains. SSTRs are highly expressed in variouscultured tumor cells and primary tumor tissues, including NETs (lung,GI, pancreatic, pituitary, medullary cancers, prostate, pancreaticlungcarcinoids, osteosarcoma, etc.) as well as non-NETs (breast, lung,colarectal, ovarian, cervical cancers, etc.) (Reubi., 2003, Endocr. Rev.24: 389-427; Volante et al., 2008, Mol. Cell. Endocrinol. 286: 219-229;and Schulz et al., 2003, Gynecol. Oncol. 89: 385-390). To date, fiveSSTR receptor subtypes have been identified (Patel et al., 1997, TrendsEndocrinol. Metab. 8: 398-405). SSTR2 in particular is expressed at ahigh concentration on many tumor cells (Volante et al., 2008, Mol. Cell.Endocrinol. 286: 219-229; and Reubi et al., 2003, Eur. J. Nucl. Med.Mol. Imaging 30: 781-793), thus making it a candidate target antigen forbispecific antibody cancer target therapeutics. In view of the highconcentration of SSTR2 expressed on various tumors, it is believed thatanti-SSTR2 antibodies are useful, for example, for localizing anti-tumortherapeutics (e.g., chemotherapeutic agents and T cells) to such SSTR2expressing tumors. For example, bispecific antibodies to SSTR2 and CD3that are capable of localizing CD3+ effector T cells to SSTR2 expressingtumors are believed to be useful cancer therapeutics. While bispecificsgenerated from antibody fragments suffer biophysical and pharmacokinetichurdles, a drawback of those built with full length antibody-likeformats is that they engage co-target antigens multivalently in theabsence of the primary target antigen, leading to nonspecific activationand potentially toxicity. The present invention solves this problem byintroducing novel bispecific antibodies directed to SSTR2 and CD3.

BRIEF SUMMARY OF THE INVENTION

Accordingly, provided herein are somatostatin receptor 2 (SSTR2) antigenbinding domains and anti-SSTR2 antibodies (e.g., bispecific antibodies).

In one aspect, provided herein are SSTR2 “bottle opener” formatantibodies that include: a) a first heavy chain that includes i) a firstvariant Fc domain; and ii) a single chain Fv region (scFv), where thescFv region includes a first variable heavy domain, a first variablelight domain and a charged scFv linker, where the charged scFv linkercovalently attaches the first variable heavy domain and the firstvariable light domain; b) a second heavy chain that includes aVH-CH1-hinge-CH2-CH3 monomer, where VH is a second variable heavy domainand CH2-CH3 is a second variant Fc domain; and c) a light chain thatincludes a second variable light domain and a light constant domain. Thesecond variant Fc domain includes amino acid substitutionsN208D/Q295E/N384D/Q418E/N421D, the first and second variant Fc domainseach include amino acid substitutions E233P/L234V/L235A/G236del/S267K;the first variant Fc domain includes amino acid substitutionsS364K/E357Q and the second variant Fc domain the amino acidsubstitutions L368D/K370S. Further, the second variable heavy domainincludes SEQ ID NO: 1071 and the second variable light domain includesSEQ ID NO: 1076, where numbering is according to the EU index as inKabat.

In certain embodiments of the SSTR2 “bottle opener” format antibodies,the scFv binds CD3. In some embodiments, the first variable heavy domainand the first variable light domain are selected from the setscomprising: SEQ ID NO: 1 and SEQ ID NO: 5; SEQ ID NO: 10 and SEQ ID NO:14; SEQ ID NO: 19 and SEQ ID NO: 23; SEQ ID NO: 28 and SEQ ID NO: 32;SEQ ID NO: 37 and SEQ ID NO: 41; and SEQ ID NO: 46 and SEQ ID NO: 50,respectively. In some embodiments, the first variable heavy domainincludes SEQ ID NO: 1 and the first variable light domain includes SEQID NO: 5.

In certain embodiments of the SSTR2 “bottle opener” format antibodies,the CH1-hinge-CH2-CH3 component of the second heavy chain includes SEQID NO: 1108, the first variant Fc domain includes SEQ ID NO: 1109 andthe constant light domain includes SEQ ID NO: 1110.

In some embodiments, the first heavy chain includes SEQ ID NO: 1080, thesecond heavy chain includes SEQ ID NO: 1070, and the light chainincludes SEQ ID NO: 1075.

In another aspect provided herein is a somatostatin receptor type 2(SSTR2) antigen binding domain, that includes a variable heavy domainhaving SEQ ID NO: 958 and a variable light domain having SEQ ID NO: 962.

In another aspect, provided herein is a nucleic acid composition thatincludes nucleic acids encoding any of the heterodimeic antibodies orantigen binding domains described herein.

In yet another aspect, provided herein is an expression vector thatincludes any of the nucleic acids described herein.

In one aspect, provided herein is a host cell transformed with any ofthe expression vectors or nucleic acids described herein.

In another aspect, provided herein is a method of making a subjectheterodimeric antibody or antigen binding domain described herein. Themethod includes a step of culturing a host cell transformed with any ofthe expression vectors or nucleic acids described herein underconditions wherein the antibody or antigen binding domain is expressed,and recovering the antibody or antigen binding domain.

In one aspect, provided herein is a method of treating cancer thatincludes administering to a patient in need thereof any one of thesubject antibodies described herein.

In some embodiments, the cancer is a neuroendocrine cancer.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A-1I depict several formats of the present invention. The first isthe “bottle opener” format, with a first and a second anti-antigenbinding domain. Additionally, mAb-Fv, mAb-scFv, Central-scFv,Central-Fv, one armed central-scFv, one scFv-mAb, scFv-mAb and a dualscFv format are all shown. For all of the scFv domains depicted, theycan be either N- to C-terminus variable heavy-(optional linker)-variablelight, or the opposite. In addition, for the one armed scFv-mAb, thescFv can be attached either to the N-terminus of a heavy chain monomeror to the N-terminus of the light chain. In certain embodiments,“Anti-antigen 1” in FIG. 1 refers to an anti-SSTR2 binding domain. Incertain embodiments “Anti-antigen 1” in FIG. 1 refers to an anti-CD3binding domain. In certain embodiments, “Anti-antigen 2” in FIG. 1refers to an anti-SSTR2 binding domain. In certain embodiments“Anti-antigen2” in FIG. 1 refers to an anti-CD3 binding domain. In someembodiments, “Anti-antigen 1” in FIG. 1 refers to an anti-SSTR2 bindingdomain and “Anti-antigen 2” in FIG. 1 refers to an anti-CD3 bindingdomain. In some embodiments, “Anti-antigen 1” in FIG. 1 refers to ananti-CD3 binding domain and “Anti-antigen 2” in FIG. 1 refers to ananti-SSTR2 binding domain.

FIG. 2 depicts the amino acid sequences for human and Cynomolgus monkey(Macaca fascicularis) SSTR2 protein.

FIG. 3A-3F depict useful pairs of heterodimerization variant sets(including skew and pI variants). On FIG. 3F, there are variants forwhich there are no corresponding “monomer 2” variants; these are pIvariants which can be used alone on either monomer, or included on theFab side of a bottle opener, for example, and an appropriate chargedscFv linker can be used on the second monomer that utilizes a scFv asthe second antigen binding domain. Suitable charged linkers are shown inFIGS. 7A and B.

FIG. 4 depicts a list of isosteric variant antibody constant regions andtheir respective substituions. pI_(−) indicates lower pI variants, whilepI_(+) indicates higher pI variants. These can be optionally andindependently combined with other heterodimerization variants of theinvention (and other variant types as well, as outlined herein).

FIG. 5 depict useful ablation variants that ablate FcγR binding(sometimes referred to as “knock outs” or “KO” variants).

FIG. 6 show two particularly useful embodiments of the invention.

FIGS. 7A-7B depict a number of charged scFv linkers that find use inincreasing or decreasing the pI of the subject heterodimeric antibodiesthat utilize one or more scFv as a component, as described herein. The(+H) positive linker finds particular use herein, particularly withanti-CD3 vl and vh sequences shown herein. A single prior art scFvlinker with a single charge is referenced as “Whitlow”, from Whitlow etal., Protein Engineering 6(8):989-995 (1993). It should be noted thatthis linker was used for reducing aggregation and enhancing proteolyticstability in scFvs.

FIG. 8 depicts various heterodimeric skewing variant amino acidsubstitutions that can be used with the heterodimeric antibodiesdescribed herein.

FIG. 9A-9E shows the sequences of several useful bottle opener formatbackbones based on human IgG1, without the Fv sequences (e.g. the scFvand the vh and vl for the Fab side). Bottle opener backbone 1 is basedon human IgG1 (356E/358M allotype), and includes theS364K/E357Q:L368D/K370S skew variants, the N208D/Q295E/N384D/Q418E/N421DpI variants on the Fab side and the E233P/L234V/L235A/G236del/S267Kablation variants on both chains. Bottle opener backbone 2 is based onhuman IgG1 (356E/358M allotype), and includes different skew variants,the N208D/Q295E/N384D/Q418E/N421D pI variants on the Fab side and theE233P/L234V/L235A/G236del/S267K ablation variants on both chains. Bottleopener backbone 3 is based on human IgG1 (356E/358M allotype), andincludes different skew variants, the N208D/Q295E/N384D/Q418E/N421D pIvariants on the Fab side and the E233P/L234V/L235A/G236del/S267Kablation variants on both chains. Bottle opener backbone 4 is based onhuman IgG1 (356E/358M allotype), and includes different skew variants,the N208D/Q295E/N384D/Q418E/N421D pI variants on the Fab side and theE233P/L234V/L235A/G236del/S267K ablation variants on both chains. Bottleopener backbone 5 is based on human IgG1 (356D/358L allotype), andincludes the S364K/E357Q:L368D/K370S skew variants, theN208D/Q295E/N384D/Q418E/N421D pI variants on the Fab side and theE233P/L234V/L235A/G236del/S267K ablation variants on both chains. Bottleopener backbone 6 is based on human IgG1 (356E/358M allotype), andincludes the S364K/E357Q:L368D/K370S skew variants,N208D/Q295E/N384D/Q418E/N421D pI variants on the Fab side and theE233P/L234V/L235A/G236del/S267K ablation variants on both chains, aswell as an N297A variant on both chains. Bottle opener backbone 7 isidentical to 6 except the mutation is N297S. Alternative formats forbottle opener backbones 6 and 7 can exclude the ablation variantsE233P/L234V/L235A/G236del/S267K in both chains. Backbone 8 is based onhuman IgG4, and includes the S364K/E357Q:L368D/K370S skew variants, theN208D/Q295E/N384D/Q418E/N421D pI variants on the Fab side and theE233P/L234V/L235A/G236del/S267K ablation variants on both chains, aswell as a S228P (EU numbering, this is S241P in Kabat) variant on bothchains that ablates Fab arm exchange as is known in the art. Alternativeformats for bottle opener backbone 8 can exclude the ablation variantsE233P/L234V/L235A/G236del/S267K in both chains Backbone 9 is based onhuman IgG2, and includes the S364K/E357Q:L368D/K370S skew variants, theN208D/Q295E/N384D/Q418E/N421D pI variants on the Fab side. Backbone 10is based on human IgG2, and includes the S364K/E357Q:L368D/K370S skewvariants, the N208D/Q295E/N384D/Q418E/N421D pI variants on the Fab sideas well as a S267K variant on both chains.

As will be appreciated by those in the art and outlined below, thesesequences can be used with any vh and vl pairs outlined herein, with onemonomer including a scFv (optionally including a charged scFv linker)and the other monomer including the Fab sequences (e.g. a vh attached tothe “Fab side heavy chain” and a vl attached to the “constant lightchain”). That is, any Fv sequences outlined herein for anti-SSTR2 andanti-CD3, whether as scFv (again, optionally with charged scFv linkers)or as Fabs, can be incorporated into these FIG. 9 backbones in anycombination. The constant light chain depicted in FIG. 9A can be usedfor all of the constructs in the figure, although the kappa constantlight chain can also be substituted.

It should be noted that these bottle opener backbones find use in theCentral-scFv format of FIG. 1F, where an additional, second Fab (vh-CH1and vl-constant light) with the same antigen binding as the first Fab isadded to the N-terminus of the scFv on the “bottle opener side”.

Included within each of these backbones are sequences that are 90, 95,98 and 99% identical (as defined herein) to the recited sequences,and/or contain from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 additional aminoacid substitutions (as compared to the “parent” of the Figure, which, aswill be appreciated by those in the art, already contain a number ofamino acid modifications as compared to the parental human IgG1 (or IgG2or IgG4, depending on the backbone). That is, the recited backbones maycontain additional amino acid modifications (generally amino acidsubstitutions) in addition to the skew, pI and ablation variantscontained within the backbones of this figure.

FIG. 10A-10D shows the sequences of a mAb-scFv backbone of use in theinvention, to which the Fv sequences of the invention are added.mAb-scFv backbone 1 is based on human IgG1 (356E/358M allotype), andincludes the S364K/E357Q:L368D/K370S skew variants, theN208D/Q295E/N384D/Q418E/N421D pI variants on the Fab side and theE233P/L234V/L235A/G236del/S267K ablation variants on both chains.Backbone 2 is based on human IgG1 (356D/358L allotype), and includes theS364K/E357Q:L368D/K370S skew variants, the N208D/Q295E/N384D/Q418E/N421DpI variants on the Fab side and the E233P/L234V/L235A/G236del/S267Kablation variants on both chains. Backbone 3 is based on human IgG1(356E/358M allotype), and includes the S364K/E357Q:L368D/K370S skewvariants, N208D/Q295E/N384D/Q418E/N421D pI variants on the Fab side andthe E233P/L234V/L235A/G236del/S267K ablation variants on both chains, aswell as an N297A variant on both chains. Backbone 4 is identical to 3except the mutation is N297S. Alternative formats for mAb-scFv backbones3 and 4 can exclude the ablation variantsE233P/L234V/L235A/G236del/S267K in both chains. Backbone 5 is based onhuman IgG4, and includes the S364K/E357Q:L368D/K370S skew variants, theN208D/Q295E/N384D/Q418E/N421D pI variants on the Fab side and theE233P/L234V/L235A/G236del/S267K ablation variants on both chains, aswell as a S228P (EU numbering, this is S241P in Kabat) variant on bothchains that ablates Fab arm exchange as is known in the art Backbone 6is based on human IgG2, and includes the S364K/E357Q:L368D/K370S skewvariants, the N208D/Q295E/N384D/Q418E/N421D pI variants on the Fab side.Backbone 7 is based on human IgG2, and includes theS364K/E357Q:L368D/K370S skew variants, the N208D/Q295E/N384D/Q418E/N421DpI variants on the Fab side as well as a S267K variant on both chains.

As will be appreciated by those in the art and outlined below, thesesequences can be used with any vh and vl pairs outlined herein, with onemonomer including both a Fab and an scFv (optionally including a chargedscFv linker) and the other monomer including the Fab sequence (e.g. a vhattached to the “Fab side heavy chain” and a vl attached to the“constant light chain”). That is, any Fv sequences outlined herein foranti-SSTR2 and anti-CD3, whether as scFv (again, optionally with chargedscFv linkers) or as Fabs, can be incorporated into this FIG. 10 backbonein any combination. The monomer 1 side is the Fab-scFv pI negative side,and includes the heterodimerization variants L368D/K370S, the isostericpI variants N208D/Q295E/N384D/Q418E/N421D, the ablation variantsE233P/L234V/L235A/G236del/S267K, (all relative to IgG1). The monomer 2side is the scFv pI positive side, and includes the heterodimerizationvariants 364K/E357Q. However, other skew variant pairs can besubstituted, particularly [S364K/E357Q:L368D/K370S];[L368D/K370S:S364K]; [L368E/K370S:S364K]; [T411T/E360E/Q362E:D401K];[L368D/K370S:S364K/E357L], [K370S:S364K/E357Q],[T366S/L368A/Y407V:T366W] and [T366S/L368A/Y407V/Y394C:T366W/S354C].

The constant light chain depicted in FIG. 10A can be used for all of theconstructs in the figure, although the kappa constant light chain canalso be substituted.

It should be noted that these mAb-scFv backbones find use in the boththe mAb-Fv format of FIG. 1H (where one monomer comprises a vl at theC-terminus and the other a vh at the C-terminus) as well as the scFv-mAbformat of FIG. 1E (with a scFv domain added to the C-terminus of one ofthe monomers).

Included within each of these backbones are sequences that are 90, 95,98 and 99% identical (as defined herein) to the recited sequences,and/or contain from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 additional aminoacid substitutions (as compared to the “parent” of the Figure, which, aswill be appreciated by those in the art, already contain a number ofamino acid modifications as compared to the parental human IgG1 (or IgG2or IgG4, depending on the backbone). That is, the recited backbones maycontain additional amino acid modifications (generally amino acidsubstitutions) in addition to the skew, pI and ablation variantscontained within the backbones of this figure.

FIGS. 11A-11G depict the amino acid sequences of exemplary subjectanti-SSTR2 antigen binding domains described herein, includinganti-SSTR2 H1.143_L1.30; anti-SSTR2 H1 L1.1; anti-SSTR2 H1.107_L1.30;anti-SSTR2 H1.107_L1.67; anti-SSTR2 H1.107 L1.108; anti-SSTR2 H1.107L1.111; anti-SSTR2 H1.107 L1.114; anti-SSTR2 H1.107 L1.102; anti-SSTR2H1.107 L1.110; anti-SSTR2 H1.125 L1.30; anti-SSTR2 H1.125_L1.67;anti-SSTR2 H1.125_L1.108; anti-SSTR2 H1.125_L1.111; anti-SSTR2H1.125_L1.114; anti-SSTR2 H1.125_L1.102; and anti-SSTR2 H1.125_L1.10.Sequences depicted include variable heavy (vh) domains and variablelight (vl) domain sequences for each antigen binding domain. For each vhsequence, vhCDR1, vhCDR2, and vhCDR3 sequences are underlined and inblue. For each vl sequence, vlCDR1, vlCDR2, and vlCDR3 sequences areunderlined and in blue. As noted herein and is true for every sequenceherein containing CDRs, the exact identification of the CDR locationsmay be slightly different depending on the numbering used as is shown inTable 1, and thus included herein are not only the CDRs that areunderlined but also CDRs included within the vh and vl domains usingother numbering systems. Furthermore, as for all the sequences in theFigures, these vh and vl sequences can be used either in a scFv formator in a Fab format.

FIGS. 12A-12F depict various anti-CD3 antigen binding domains (e.g.,anti-CD3 scFvs) that can be used in the subject antibodies providedherein. The CDRs are underlined, the scFv linker is double underlined(in the sequences, the scFv linker is a positively charged scFv (GKPGS)4linker, although as will be appreciated by those in the art, this linkercan be replaced by other linkers, including uncharged or negativelycharged linkers, some of which are depicted in FIG. 7. As above, thenaming convention illustrates the orientation of the scFv from N- toC-terminus; in the sequences listed in this figure, they are alloriented as vh-scFv linker-vl (from N- to C-terminus), although thesesequences may also be used in the opposite orientation, (from N- toC-terminus) vl-linker-vh. As noted herein and is true for every sequenceherein containing CDRs, the exact identification of the CDR locationsmay be slightly different depending on the numbering used as is shown inTable 1, and thus included herein are not only the CDRs that areunderlined but also CDRs included within the vh and vl domains usingother numbering systems. Furthermore, as for all the sequences in theFigures, these vh and vl sequences can be used either in a scFv formator in a Fab format.

FIG. 12A depicts the sequences of the “High CD3” anti-CD3_H1.30_L1.47construct, including the variable heavy and light domains (CDRsunderlined), as well as the individual vl and vhCDRs, as well as an scFvconstruct with a charged linker (double underlined). As is true of allthe sequences depicted in the Figures, this charged linker may bereplaced by an uncharged linker or a different charged linker, asneeded.

FIG. 12B depicts the sequences of the “High-Int #1” Anti-CD3_H1.32_L1.47construct, including the variable heavy and light domains (CDRsunderlined), as well as the individual vl and vhCDRs, as well as an scFvconstruct with a charged linker (double underlined). As is true of allthe sequences depicted in the Figures, this charged linker may bereplaced by an uncharged linker or a different charged linker, asneeded.

FIG. 12C depicts the sequences of the “High-Int #2” Anti-CD3 H1.89_L1.47construct, including the variable heavy and light domains (CDRsunderlined), as well as the individual vl and vhCDRs, as well as an scFvconstruct with a charged linker (double underlined). As is true of allthe sequences depicted in the Figures, this charged linker may bereplaced by an uncharged linker or a different charged linker, asneeded.

FIG. 12D depicts the sequences of the “High-Int #3” Anti-CD3_H1.90_L1.47construct, including the variable heavy and light domains (CDRsunderlined), as well as the individual vl and vhCDRs, as well as an scFvconstruct with a charged linker (double underlined). As is true of allthe sequences depicted in the Figures, this charged linker may bereplaced by an uncharged linker or a different charged linker, asneeded.

FIG. 12E depicts the sequences of the “Int” Anti-CD3_H1.33_L1.47construct, including the variable heavy and light domains (CDRsunderlined), as well as the individual vl and vhCDRs, as well as an scFvconstruct with a charged linker (double underlined). As is true of allthe sequences depicted in the Figures, this charged linker may bereplaced by an uncharged linker or a different charged linker, asneeded.

FIG. 12F depicts the sequences of the “Low” Anti-CD3_H1.31_L1.47construct, including the variable heavy and light domains (CDRsunderlined), as well as the individual vl and vhCDRs, as well as an scFvconstruct with a charged linker (double underlined). As is true of allthe sequences depicted in the Figures, this charged linker may bereplaced by an uncharged linker or a different charged linker, asneeded.

FIGS. 13A-13Z depict amino acid sequences of stability-optimized,humanized anti-CD3 variant scFvs variants that can be used with thesubject bispecific antibodies described herein (e.g, anti-SSTR2×anti-CD3“bottle opener” antibodies). CDRs are underlined. For each heavychain/light chain combination, four sequences are listed: (i) scFv withC-terminal 6×His tag, (ii) scFv alone, (iii) VH alone, (iv) VL alone. Asnoted herein and is true for every sequence herein containing CDRs, theexact identification of the CDR locations may be slightly differentdepending on the numbering used as is shown in Table 1, and thusincluded herein are not only the CDRs that are underlined but also CDRsincluded within the vh and vl domains using other numbering systems.Furthermore, as for all the sequences in the Figures, these vh and vlsequences can be used either in a scFv format or in a Fab format.

FIGS. 14A-14B depict the amino acid sequences of an exemplaryanti-SSTR2×anti-CD3 “bottle-opener” bispecific antibody describedherein, XENP018087 (SSTR2 H1.143_L1.30 and CD3 H1.30_L1.47). For theSSTR2 Fab-Fc heavy chain sequence, vhCDRs1-3 are underlined and in blueand the border between the variable heavy domain and CH1-hinge-CH2-CH3is indicated by by “/”. For the CD3 scFv-Fc heavy chain sequence,borders between various domains are indicated using “/” and are asfollows: scFv variable heavy chain domain/scFv linker/scFv light chaindomain/Fc domain. vhCDRs1-3 and vlCDRs1-3 are underlined in blue. Foreach scFv-Fc domain, the vhCDR1-3 and vlCDR1-3 sequences are underlinedand in blue. For the CD3 light chain sequence, vlCDRs1-3 are underlinedand in blue and the border between the variable light chain domain andthe light chain constant domain is indicated by “/”. The charged linkerdepicted is (GKPGS)4, although other charged or uncharged linkers can beused, such as those depicted in FIGS. 7A and B. In addition, eachsequence outlined herein can include or exclude the M428L/N434S variantsin one or preferably both Fc domains, which results in longer half-lifein serum.

FIGS. 15A-15R depict the amino acid sequences of additional exemplaryanti-SSTR2×anti-CD3 “bottle-opener” bispecific antibody describedherein, including XENP018907(FIGS. 15 A and B, SSTR2 H1.143_L1.30 andCD3 H1.32_L1.47). For the SSTR2 Fab-Fc heavy chain sequence, vhCDRs1-3are underlined and in blue and the border between the variable heavydomain and CH1-hinge-CH2-CH3 is indicated by by “/”. For the CD3 scFv-Fcheavy chain sequence, borders between various domains are indicatedusing “/” and are as follows: scFv variable heavy chain domain/scFvlinker/scFv light chain domain/Fc domain. vhCDRs1-3 and vlCDRs1-3 areunderlined in blue. For each scFv-Fc domain, the vhCDR1-3 and vlCDR1-3sequences are underlined and in blue. For the CD3 light chain sequence,vlCDRs1-3 are underlined and in blue and the border between the variablelight chain domain and the light chain constant domain is indicated by“/”. The charged linker depicted is (GKPGS)4, although other charged oruncharged linkers can be used, such as those depicted in FIGS. 7A and B.In addition, each sequence outlined herein can include or exclude theM428L/N434S variants in one or preferably both Fc domains, which resultsin longer half-life in serum.

FIGS. 16A-16C depict matrices of possible combinations for exemplarybispecific anti-SSTR2×anti-CD3 antibodies described herein. An “A” meansthat the CDRs of the referenced CD3 binding domain sequences at the topof the matrix can be combined with the CDRs of the SSTR2 binding domainsequences listed on the left hand side of the matrix. For example, withrespect to “Anti-SSTR2 H1.143_L1.30” and “Anti-CD3 H1.30_L1.47”, “A”indicates a bispecific antibody that includes a) a CD3 binding domainhaving vhCDRs from the variable heavy chain CD3 H1.30 sequence and thevlCDRs from the variable light chain CD3 L1.47 sequence, and b) an SSTR2binding domain having the vhCDRs from the SSTR2 H1.143 sequence and thevlCDRs from the SSTR2 L1.30 sequence. A “B” means that the CDRs from theCD3 binding domain constructs can be combined with the variable heavyand light domains from the SSTR2 binding domain constructs. For example,with respect to “Anti-SSTR2 H1.143_L1.30” and “Anti-CD3 H1.30_L1.47”,“B” indicates a bispecific antibody that includes a) a CD3 bindingdomain having the vhCDRs from the variable heavy chain CD3 H1.30sequence and the vlCDRs from the variable light chain of CD3 L1.47sequence, and b) a SSTR2 binding domain having the variable heavy domainSSTR2 H1.143 sequence and the variable light domain SSTR2 L1.30sequence. A “C” indicates a bispecific antibody that includes a) a CD3binding domain having a variable heavy domain and variable light domainfrom the anti-CD3 sequences, and b) a SSTR2 binding domain with the CDRsof the anti-SSTR2 sequences. A “D” indicates a bispecific antibody thatincludes an SSTR2 binding domain having the variable heavy and variablelight chain of the indicated anti-SSTR2 sequence and a CD3 bindingdomain having the variable heavy and variable light chain of theindicated anti-CD3 sequence. An “E” indicates a bispecific antibody thatincludes an scFv, where the scFv of the CD3 is used with the CDRs of theSSTR2. An “F” indicates a bispecific antibody that includes an scFv,where the scFv of the CD3 is used with the variable heavy and variablelight domains of the SSTR2 antigen binding domain. All of thesecombinations can be done in bottle opener formats, for example with anyof the backbone formats shown in FIG. 9, or in alternative formats, suchas mAb-Fv, mAb-scFv, Central-scFv, Central-Fv or dual scFv formats ofFIG. 1, including the format backbones shown in FIG. 26. For example,“A”s (CD3 CDRs and SSTR2 CDRs) can be added to bottle opener sequences,including those of FIG. 9 or inclusive of different heterodimerizationvariants, or into a mAb-scFv backbone of FIG. 10, a central-scFv, amAb-Fv format or a central-Fv format. In general, however, formats thatwould include bivalent binding of CD3 are disfavored.

FIGS. 16D-16F depict matrices of possible combinations for exemplarybispecific anti-SSTR2×anti-CD3 bottle opener format combinationsdescribed herein. In these matrices, the anti-CD3 scFvs are listed inthe X axis and the anti-SSTR2 Fabs are listed on the Y axis. An “A”means that the CDRs of the referenced CD3 binding domain sequences atthe top of the matrix can be combined with the CDRs of the SSTR2 bindingdomain sequences listed on the left hand side of the matrix. Forexample, with respect to “Anti-SSTR2 H1.143_L1.30” and “Anti-CD3H1.30_L1.47”, “A” indicates a bispecific bottle opener format antibodythat includes a) an anti-CD3 scFV having vhCDRs from the variable heavychain CD3 H1.30 sequence and the vlCDRs from the variable light chainCD3 L1.47 sequence, and b) an anti-SSTR2 Fab having the vhCDRs from theSSTR2 H1.143 sequence and the vlCDRs from the SSTR2 L1.30 sequence. A“B” means that the CDRs from the CD3 binding domain constructs can becombined with the variable heavy and light domains from the SSTR2binding domain constructs. For example, with respect to “Anti-SSTR2H1.143_L1.30” and “Anti-CD3 H1.30_L1.47”, “B” indicates a bispecificbottle opener antibody that includes a) a anti-CD3 scFv having thevhCDRs from the variable heavy chain CD3 H1.30 sequence and the vlCDRsfrom the variable light chain of CD3 L1.47 sequence, and b) ananti-SSTR2 Fab having the variable heavy domain SSTR2 H1.143 sequenceand the variable light domain SSTR2 L1.30 sequence. A “C” indicates abispecific bottle opener antibody that includes a) anti-CD3 scFv havinga variable heavy domain and variable light domain from the anti-CD3sequences, and b) a SSTR2 Fab with the CDRs of the anti-SSTR2 sequences.A “D” indicates a bispecific bottle opener antibody that includes an ananti-SSTR2 Fab having the variable heavy and variable light chain of theindicated SSTR2 sequence and an anti-CD3 scFv having the variable heavyand variable light chain of the indicated anti-CD3 sequence.

FIGS. 17A-17P depict cell surface binding assays of exemplary anti-SSTR2antibodies and anti-SSTR2×anti-CD3 bispecific antibodies using humanSSTR2 transfected CHO cells. Binding was measured by flow cytometryusing phycoerythrin (PE) labeled secondary antibody.

FIGS. 18A-18D depict results of redirected T cell cytotoxicity (RTCC)assay, using anti-SSTR2×anti-CD3 bispecifics and human SSTR2 transfectedCHO cells.

FIGS. 19A-19C depict the results of redirected T cell cytotoxicity(RTCC) assay, using anti-SSTR2×anti-CD3 bispecifics with TT cells (humanthyroid medullary carcinoma cell line, FIGS. 19A-19C).

FIGS. 20A-20B depict a study of the effects of anti-SSTR2×anti-CD3bispecific antibodies on CD4⁺ and CD8⁺ T cell activation (FIG. 20A) andCD4⁺ and CD8⁺ T cell distribution (FIG. 20B) in cynomolgus monkeys.

FIGS. 21A-21D depict additional studies of the effects ofanti-SSTR2×anti-CD3 bispecific antibodies on CD4⁺ and CD8⁺ T cellactivation (FIG. 21A) and CD4++ and CD8++ T cell distribution (FIG. 21B)in cynomolgus monkeys. In addition, a glucose tolerance test (GTT) wasconducted (FIGS. 21C and 21D) to assess the ability of the testedsubjects to breakdown glucose.

FIGS. 22A-22F depict additional studies of an exemplaryanti-SSTR2×anti-CD3 bispecific antibody on CD4⁺ and CD8⁺ T cellactivation (FIGS. 22A and B), CD4⁺ and CD8⁺ T cell distribution (FIGS.22C and D) and serum levels of serum IL-6 and TNFα (FIGS. 22E and F).

FIGS. 23A-23C depict cell surface binding assays of XmAb18087 andXENP13245 on human SSTR2-transfected CHO cells (FIG. 22A), cynoSSTR2-transfected CHO cells (FIG. 22B), and untransfected parental CHOcells (FIG. 22C).

FIGS. 24A-24C depict the results of a redirected T cell cytotoxicity(RTCC) assay, using XmAb18087 (squares) and XENP13245 (circles) withhuman SSTR2-transfected CHO cells (FIG. 24A), TT cells (human thyroidmedullary carcinoma cell line, FIG. 28B) or A548 cells (lungadenocarcinoma cell line, FIG. 24C).

FIG. 25 depicts the results of a redirected T cell cytotoxicity (RTCC)assay, using anti-SSTR2×anti-CD3 bispecific and controls anti-SSTR2 mAband anti-RSV×anti-CD3 with TT cells (human thyroid medullary carcinomacell line) or A548 cells (lung carcinoma).

FIGS. 26A-26B depicts upregulation of CD69 on CD4⁺ and CD8⁺ T cellsincubated with human SSTR2 transfected CHO cells (FIG. 29A) and TT cells(FIG. 29B) after 24 h for the experiment described in FIG. 2. Filleddata points show CD69 MFI on CD8⁺ T cells and empty data points showCD69 MFI on CD4⁺ T cells.

FIG. 27 depicts the design of mouse study to examine anti-tumor activityof XmAb18087.

FIG. 28A-28B depicts tumor size measured by IVIS® as a function of timeand treatment.

FIG. 29 depicts IVIS® bioluminescent images (Day 28 post dose #1).

FIGS. 30A-30B depict a study of the effects of XmAb18087 on CD4⁺ (FIG.30A) and CD8⁺ (FIG. 30B) T cell distribution in cynomolgus monkeys.

FIGS. 31A-31B depict a study of the effects of XmAb18087 on CD4⁺ (FIG.31A) and CD8⁺ (FIG. 31B) T cell activation in cynomolgus monkeys.

FIGS. 32A-32B depicts the effect of XmAb18087 on the level of serum IL-6and TNF in cynomolgus monkeys.

FIG. 33 depict tumor size in NSG mice engrafted with A549-RedFLuc tumorcells and human PBMCs as measured by IVIS® as a function of time andtreatment using various concentrations of XmAb18087.

DETAILED DESCRIPTION OF THE INVENTION A. Incorporation of Materials

Figures and Legends

All the figures and accompanying legends of U.S. Ser. Nos. 62/481,065,62/397,322, 62/355,821 and 62/355,820 are expressly and independentlyincorporated by reference herein in their entirety, particularly for theamino acid sequences depicted therein.

Sequences

Reference is made to the accompanying sequence listing as follows.Anti-SSTR2 sequences suitable for use as ABDs include SEQ ID NOs:958-1069 (FIG. 11) and the variable heavy domain, the variable lightdomain, and CDRs of the anti-SSTR2 heavy chain and light chain sequencesof SEQ ID NOs: 58 to 659. Anti-CD3 sequences suitable for use as ABDsinclude the variable heavy domain, the variable light domain, and CDRsincluded in SEQ ID NOs: 1-54 (FIG. 12) and SEQ ID NOs: 835 to 938. Thevariable heavy domain, the variable light domain, and CDRs can beincluded in scFv or Fv formats of the subject antibodies and antigenbinding domains described herein.

Sequences of exemplary bispecific SSTR2×CD3 antibodies are included inSEQ ID NO: 1070 to 1088 (FIG. 14); and SEQ ID NOs: 1089 to 1107 and 660to 806 (FIG. 15).

B. Overview

Provided herein are anti-SSTR2 antibodies that are useful for thetreatment of cancers. As SSTR2 is high expressed in neuroendocrinetumors (NETs, e.g., lung, GI, pancreatic, pituitary, medullary cancers,prostate, pancreatic lungcarcinoids, osteosarcoma, etc.) as well asnon-NETs (breast, lung, colarectal, ovarian, cervial cancers, etc.), itis believed that anti-SSTR2 antibodies are useful for localizinganti-tumor therapeutics (e.g., chemotherapeutic agents and T cells) tosuch SSTR2 expressing tumors. In particular, provided herein areanti-CD3, anti-SSTR2 bispecific antibodies. Such antibodies are used todirect CD3+ effector T cells to SSTR2+ tumors, thereby allowing the CD3+effector T cells to attack and lyse the SSTR2+ tumors.

Anti-bispecific antibodies that co-engage CD3 and a tumor antigen targethave been designed and used to redirect T cells to attack and lysetargeted tumor cells. Examples include the BiTE and DART formats, whichmonovalently engage CD3 and a tumor antigen. While the CD3-targetingapproach has shown considerable promise, a common side effect of suchtherapies is the associated production of cytokines, often leading totoxic cytokine release syndrome. Because the anti-CD3 binding domain ofthe bispecific antibody engages all T cells, the high cytokine-producingCD4 T cell subset is recruited. Moreover, the CD4 T cell subset includesregulatory T cells, whose recruitment and expansion can potentially leadto immune suppression and have a negative impact on long-term tumorsuppression. In addition, these formats do not contain Fc domains andshow very short serum half-lives in patients.

While the CD3-targeting approach has shown considerable promise, acommon side effect of such therapies is the associated production ofcytokines, often leading to toxic cytokine release syndrome. Because theanti-CD3 binding domain of the bispecific antibody engages all T cells,the high cytokine-producing CD4 T cell subset is recruited. Moreover,the CD4 T cell subset includes regulatory T cells, whose recruitment andexpansion can potentially lead to immune suppression and have a negativeimpact on long-term tumor suppression. One such possible way to reducecytokine production and possibly reduce the activation of CD4 T cells isby reducing the affinity of the anti-CD3 domain for CD3.

Accordingly, in some embodiments the present invention provides antibodyconstructs comprising anti-CD3 antigen binding domains that are “strong”or “high affinity” binders to CD3 (e.g. one example are heavy and lightvariable domains depicted as H1.30_L1.47 (optionally including a chargedlinker as appropriate)) and also bind to SSTR2. In other embodiments,the present invention provides antibody constructs comprising anti-CD3antigen binding domains that are “lite” or “lower affinity” binders toCD3. Additional embodiments provides antibody constructs comprisinganti-CD3 antigen binding domains that have intermediate or “medium”affinity to CD3 that also bind to CD38. Affinity is generally measuredusing a Biacore assay.

It should be appreciated that the “high, medium, low” anti-CD3 sequencesof the present invention can be used in a variety of heterodimerizationformats. While the majority of the disclosure herein uses the “bottleopener” format of heterodimers, these variable heavy and lightsequences, as well as the scFv sequences (and Fab sequences comprisingthese variable heavy and light sequences) can be used in other formats,such as those depicted in FIG. 2 of WO Publication No. 2014/145806, theFigures, formats and legend of which is expressly incorporated herein byreference.

Accordingly, in one aspect, provided herein are heterodimeric antibodiesthat bind to two different antigens, e.g the antibodies are“bispecific”, in that they bind two different target antigens, generallySSTR2 as described below. These heterodimeric antibodies can bind thesetarget antigens either monovalently (e.g. there is a single antigenbinding domain such as a variable heavy and variable light domain pair)or bivalently (there are two antigen binding domains that eachindependently bind the antigen). The heterodimeric antibodies providedherein are based on the use different monomers which contain amino acidsubstitutions that “skew” formation of heterodimers over homodimers, asis more fully outlined below, coupled with “pI variants” that allowsimple purification of the heterodimers away from the homodimers, as issimilarly outlined below. The heterodimeric bispecific antibodiesprovided generally rely on the use of engineered or variant Fc domainsthat can self-assemble in production cells to produce heterodimericproteins, and methods to generate and purify such heterodimericproteins.

C. Nomenclature

The bispecific antibodies of the invention are listed in severaldifferent formats. Each polypeptide is given a unique “XENP” number,although as will be appreciated in the art, a longer sequence mightcontain a shorter one. For example, the heavy chain of the scFv sidemonomer of a bottle opener format for a given sequence will have a firstXENP number, while the scFv domain will have a different XENP number.Some molecules have three polypeptides, so the XENP number, with thecomponents, is used as a name. Thus, the molecule XENP18087, which is inbottle opener format, comprises three sequences: “XENP 18087 HC-Fab”(FIG. 14A, termed “SSTR2 Fab-Fc Heavy Chain), “XENP18087 HC-scFv” (FIG.14B, termed “CD3 scFv-Fc Heavy Chain”) and “XENP 18087 LC” (FIG. 14A,termed “SSTR2 Light Chain”) or equivalents, although one of skill in theart would be able to identify these easily through sequence alignment.These XENP numbers are in the sequence listing as well as identifiers,and used in the Figures. In addition, one molecule, comprising the threecomponents, gives rise to multiple sequence identifiers. For example,the listing of the Fab monomer has the full length sequence, thevariable heavy sequence and the three CDRs of the variable heavysequence; the light chain has a full length sequence, a variable lightsequence and the three CDRs of the variable light sequence; and thescFv-Fc domain has a full length sequence, an scFv sequence, a variablelight sequence, 3 light CDRs, a scFv linker, a variable heavy sequenceand 3 heavy CDRs; note that all molecules herein with a scFv domain usea single charged scFv linker (+H), although others can be used. Inaddition, the naming nomenclature of particular variable domains uses a“Hx.xx_Ly.yy” type of format, with the numbers being unique identifiersto particular variable chain sequences. Thus, the variable domain of theFab side of XENP18087 is “H1.143 L1.30”, which indicates that thevariable heavy domain, H1.143, was combined with the light domain L1.30.In the case that these sequences are used as scFvs, the designation“H1.143_L1.30”, indicates that the variable heavy domain, H1.143, wascombined with the light domain, L1.30, and is in vh-linker-vlorientation, from N- to C-terminus. This molecule with the identicalsequences of the heavy and light variable domains but in the reverseorder would be named “L1.30_H1.143”. Similarly, different constructs may“mix and match” the heavy and light chains as will be evident from thesequence listing and the Figures.

D. Definitions

In order that the application may be more completely understood, severaldefinitions are set forth below. Such definitions are meant to encompassgrammatical equivalents.

By “ablation” herein is meant a decrease or removal of activity. Thusfor example, “ablating FcγR binding” means the Fc region amino acidvariant has less than 50% starting binding as compared to an Fc regionnot containing the specific variant, with more than 70-80-90-95-98% lossof activity being preferred, and in general, with the activity beingbelow the level of detectable binding in a Biacore, SPR or BLI assay. Ofparticular use in the ablation of FcγR binding are those shown in FIG.5, which generally are added to both monomers.

By “ADCC” or “antibody dependent cell-mediated cytotoxicity” as usedherein is meant the cell-mediated reaction wherein nonspecific cytotoxiccells that express FcγRs recognize bound antibody on a target cell andsubsequently cause lysis of the target cell. ADCC is correlated withbinding to FcγRIIIa; increased binding to FcγRIIIa leads to an increasein ADCC activity.

By “ADCP” or antibody dependent cell-mediated phagocytosis as usedherein is meant the cell-mediated reaction wherein nonspecificphagocytic cells that express FcγRs recognize bound antibody on a targetcell and subsequently cause phagocytosis of the target cell.

By “antigen binding domain” or “ABD” herein is meant a set of sixComplementary Determining Regions (CDRs) that, when present as part of apolypeptide sequence, specifically binds a target antigen as discussedherein. Thus, a “checkpoint antigen binding domain” binds a targetcheckpoint antigen as outlined herein. As is known in the art, theseCDRs are generally present as a first set of variable heavy CDRs (vhCDRsor VHCDRs) and a second set of variable light CDRs (vlCDRs or VLCDRs),each comprising three CDRs: vhCDR1, vhCDR2, vhCDR3 for the heavy chainand vlCDR1, vlCDR2 and vlCDR3 for the light. The CDRs are present in thevariable heavy and variable light domains, respectively, and togetherform an Fv region. (See Table 1 and related discussion above for CDRnumbering schemes). Thus, in some cases, the six CDRs of the antigenbinding domain are contributed by a variable heavy and a variable lightdomain. In a “Fab” format, the set of 6 CDRs are contributed by twodifferent polypeptide sequences, the variable heavy domain (vh or VH;containing the vhCDR1, vhCDR2 and vhCDR3) and the variable light domain(vl or VL; containing the vlCDR1, vlCDR2 and vlCDR3), with theC-terminus of the vh domain being attached to the N-terminus of the CH1domain of the heavy chain and the C-terminus of the vl domain beingattached to the N-terminus of the constant light domain (and thusforming the light chain). In a scFv format, the vh and vl domains arecovalently attached, generally through the use of a linker (a “scFvlinker”) as outlined herein, into a single polypeptide sequence, whichcan be either (starting from the N-terminus) vh-linker-vl orvl-linker-vh, with the former being generally preferred (includingoptional domain linkers on each side, depending on the format used (e.g.from FIG. 1). In general, the C-terminus of the scFv domain is attachedto the N-terminus of the hinge in the second monomer.

By “modification” herein is meant an amino acid substitution, insertion,and/or deletion in a polypeptide sequence or an alteration to a moietychemically linked to a protein. For example, a modification may be analtered carbohydrate or PEG structure attached to a protein. By “aminoacid modification” herein is meant an amino acid substitution,insertion, and/or deletion in a polypeptide sequence. For clarity,unless otherwise noted, the amino acid modification is always to anamino acid coded for by DNA, e.g. the 20 amino acids that have codons inDNA and RNA.

By “amino acid substitution” or “substitution” herein is meant thereplacement of an amino acid at a particular position in a parentpolypeptide sequence with a different amino acid. In particular, in someembodiments, the substitution is to an amino acid that is not naturallyoccurring at the particular position, either not naturally occurringwithin the organism or in any organism. For example, the substitutionE272Y refers to a variant polypeptide, in this case an Fc variant, inwhich the glutamic acid at position 272 is replaced with tyrosine. Forclarity, a protein which has been engineered to change the nucleic acidcoding sequence but not change the starting amino acid (for exampleexchanging CGG (encoding arginine) to CGA (still encoding arginine) toincrease host organism expression levels) is not an “amino acidsubstitution”; that is, despite the creation of a new gene encoding thesame protein, if the protein has the same amino acid at the particularposition that it started with, it is not an amino acid substitution.

By “amino acid insertion” or “insertion” as used herein is meant theaddition of an amino acid sequence at a particular position in a parentpolypeptide sequence. For example, −233E or 233E designates an insertionof glutamic acid after position 233 and before position 234.Additionally, −233ADE or A233ADE designates an insertion of AlaAspGluafter position 233 and before position 234.

By “amino acid deletion” or “deletion” as used herein is meant theremoval of an amino acid sequence at a particular position in a parentpolypeptide sequence. For example, E233- or E233#, E233( ) or E233deldesignates a deletion of glutamic acid at position 233. Additionally,EDA233- or EDA233# designates a deletion of the sequence GluAspAla thatbegins at position 233.

By “variant protein” or “protein variant”, or “variant” as used hereinis meant a protein that differs from that of a parent protein by virtueof at least one amino acid modification. The protein variant has atleast one amino acid modification compared to the parent protein, yetnot so many that the variant protein will not align with the parentalprotein using an alignment program such as that described below. Ingeneral, variant proteins (such as variant Fc domains, etc., outlinedherein, are generally at least 75, 80, 85, 90, 91, 92, 93, 94, 95, 96,97, 98 or 99% identical to the parent protein, using the alignmentprograms described below, such as BLAST.

As described below, in some embodiments the parent polypeptide, forexample an Fc parent polypeptide, is a human wild type sequence, such asthe heavy constant domain or Fc region from IgG1, IgG2, IgG3 or IgG4,although human sequences with variants can also serve as “parentpolypeptides”, for example the IgG1/2 hybrid of US Publication2006/0134105 can be included. The protein variant sequence herein willpreferably possess at least about 80% identity with a parent proteinsequence, and most preferably at least about 90% identity, morepreferably at least about 95-98-99% identity. Accordingly, by “antibodyvariant” or “variant antibody” as used herein is meant an antibody thatdiffers from a parent antibody by virtue of at least one amino acidmodification, “IgG variant” or “variant IgG” as used herein is meant anantibody that differs from a parent IgG (again, in many cases, from ahuman IgG sequence) by virtue of at least one amino acid modification,and “immunoglobulin variant” or “variant immunoglobulin” as used hereinis meant an immunoglobulin sequence that differs from that of a parentimmunoglobulin sequence by virtue of at least one amino acidmodification. “Fc variant” or “variant Fc” as used herein is meant aprotein comprising an amino acid modification in an Fc domain ascompared to an Fc domain of human IgG1, IgG2 or IgG4.

The Fc variants of the present invention are defined according to theamino acid modifications that compose them. Thus, for example, N434S or434S is an Fc variant with the substitution serine at position 434relative to the parent Fc polypeptide, wherein the numbering isaccording to the EU index. Likewise, M428L/N434S defines an Fc variantwith the substitutions M428L and N434S relative to the parent Fcpolypeptide. The identity of the WT amino acid may be unspecified, inwhich case the aforementioned variant is referred to as 428L/434S. It isnoted that the order in which substitutions are provided is arbitrary,that is to say that, for example, N434S/M428L is the same Fc variant asM428L/N434S, and so on. For all positions discussed in the presentinvention that relate to antibodies, unless otherwise noted, amino acidposition numbering is according to the EU index. The EU index or EUindex as in Kabat or EU numbering scheme refers to the numbering of theEU antibody. Kabat et al. collected numerous primary sequences of thevariable regions of heavy chains and light chains. Based on the degreeof conservation of the sequences, they classified individual primarysequences into the CDR and the framework and made a list thereof (seeSEQUENCES OF IMMUNOLOGICAL INTEREST, 5th edition, NIH publication, No.91-3242, E.A. Kabat et al., entirely incorporated by reference). Seealso Edelman et al., 1969, Proc Natl Acad Sci USA 63:78-85, herebyentirely incorporated by reference. The modification can be an addition,deletion, or substitution.

By “protein” herein is meant at least two covalently attached aminoacids, which includes proteins, polypeptides, oligopeptides andpeptides. In addition, polypeptides that make up the antibodies of theinvention may include synthetic derivatization of one or more sidechains or termini, glycosylation, PEGylation, circular permutation,cyclization, linkers to other molecules, fusion to proteins or proteindomains, and addition of peptide tags or labels.

By “residue” as used herein is meant a position in a protein and itsassociated amino acid identity. For example, Asparagine 297 (alsoreferred to as Asn297 or N297) is a residue at position 297 in the humanantibody IgG1.

By “Fab” or “Fab region” as used herein is meant the polypeptide thatcomprises the VH, CH1, VL, and CL immunoglobulin domains, generally ontwo different polypeptide chains (e.g. VH-CH1 on one chain and VL-CL onthe other). Fab may refer to this region in isolation, or this region inthe context of a bispecific antibody of the invention. In the context ofa Fab, the Fab comprises an Fv region in addition to the CH1 and CLdomains.

By “Fv” or “Fv fragment” or “Fv region” as used herein is meant apolypeptide that comprises the VL and VH domains of an ABD. Fv regionscan be formatted as both Fabs (as discussed above, generally twodifferent polypeptides that also include the constant regions asoutlined above) and scFvs, where the vl and vh domains are combined(generally with a linker as discussed herein) to form an scFv.

By “single chain Fv” or “scFv” herein is meant a variable heavy domaincovalently attached to a variable light domain, generally using a scFvlinker as discussed herein, to form a scFv or scFv domain. A scFv domaincan be in either orientation from N- to C-terminus (vh-linker-vl orvl-linker-vh). In the sequences depicted in the sequence listing and inthe figures, the order of the vh and vl domain is indicated in the name,e.g. H.X_L.Y means N- to C-terminal is vh-linker-vl, and L.Y_H.X isvl-linker-vh.

By “IgG subclass modification” or “isotype modification” as used hereinis meant an amino acid modification that converts one amino acid of oneIgG isotype to the corresponding amino acid in a different, aligned IgGisotype. For example, because IgG1 comprises a tyrosine and IgG2 aphenylalanine at EU position 296, a F296Y substitution in IgG2 isconsidered an IgG subclass modification.

By “non-naturally occurring modification” as used herein is meant anamino acid modification that is not isotypic. For example, because noneof the human IgGs comprise a serine at position 434, the substitution434S in IgG1, IgG2, IgG3, or IgG4 (or hybrids thereof) is considered anon-naturally occurring modification.

By “amino acid” and “amino acid identity” as used herein is meant one ofthe 20 naturally occurring amino acids that are coded for by DNA andRNA.

By “effector function” as used herein is meant a biochemical event thatresults from the interaction of an antibody Fc region with an Fcreceptor or ligand. Effector functions include but are not limited toADCC, ADCP, and CDC.

By “IgG Fc ligand” as used herein is meant a molecule, preferably apolypeptide, from any organism that binds to the Fc region of an IgGantibody to form an Fc/Fc ligand complex. Fc ligands include but are notlimited to FcγRIs, FcγRIIs, FcγRIIIs, FcRn, C1q, C3, mannan bindinglectin, mannose receptor, staphylococcal protein A, streptococcalprotein G, and viral FcγR. Fc ligands also include Fc receptor homologs(FcRH), which are a family of Fc receptors that are homologous to theFcγRs (Davis et al., 2002, Immunological Reviews 190:123-136, entirelyincorporated by reference). Fc ligands may include undiscoveredmolecules that bind Fc. Particular IgG Fc ligands are FcRn and Fc gammareceptors. By “Fc ligand” as used herein is meant a molecule, preferablya polypeptide, from any organism that binds to the Fc region of anantibody to form an Fc/Fc ligand complex.

By “Fc gamma receptor”, “FcγR” or “FcgammaR” as used herein is meant anymember of the family of proteins that bind the IgG antibody Fc regionand is encoded by an FcγR gene. In humans this family includes but isnot limited to FcγRI (CD64), including isoforms FcγRIa, FcγRIb, andFcγRIc; FcγRII (CD32), including isoforms FcγRIIa (including allotypesH131 and R131), FcγRIIb (including FcγRIIb-1 and FcγRIIb-2), andFcγRIIc; and FcγRIII (CD16), including isoforms FcγRIIIa (includingallotypes V158 and F 158) and FcγRIIIb (including allotypes FcγRIIb-NA1and FcγRIIb-NA2) (Jefferis et al., 2002, Immunol Lett 82:57-65, entirelyincorporated by reference), as well as any undiscovered human FcγRs orFcγR isoforms or allotypes. An FcγR may be from any organism, includingbut not limited to humans, mice, rats, rabbits, and monkeys. Mouse FcγRsinclude but are not limited to FcγRI (CD64), FcγRII (CD32), FcγRIII(CD16), and FcγRIII-2 (CD16-2), as well as any undiscovered mouse FcγRsor FcγR isoforms or allotypes.

By “FcRn” or “neonatal Fc Receptor” as used herein is meant a proteinthat binds the IgG antibody Fc region and is encoded at least in part byan FcRn gene. The FcRn may be from any organism, including but notlimited to humans, mice, rats, rabbits, and monkeys. As is known in theart, the functional FcRn protein comprises two polypeptides, oftenreferred to as the heavy chain and light chain. The light chain isbeta-2-microglobulin and the heavy chain is encoded by the FcRn gene.Unless otherwise noted herein, FcRn or an FcRn protein refers to thecomplex of FcRn heavy chain with beta-2-microglobulin. A variety of FcRnvariants used to increase binding to the FcRn receptor, and in somecases, to increase serum half-life. An “FcRn variant” is one thatincreases binding to the FcRn receptor, and suitable FcRn variants areshown below.

By “parent polypeptide” as used herein is meant a starting polypeptidethat is subsequently modified to generate a variant. The parentpolypeptide may be a naturally occurring polypeptide, or a variant orengineered version of a naturally occurring polypeptide. Accordingly, by“parent immunoglobulin” as used herein is meant an unmodifiedimmunoglobulin polypeptide that is modified to generate a variant, andby “parent antibody” as used herein is meant an unmodified antibody thatis modified to generate a variant antibody. It should be noted that“parent antibody” includes known commercial, recombinantly producedantibodies as outlined below. In this context, a “parent Fc domain” willbe relative to the recited variant; thus, a “variant human IgG1 Fcdomain” is compared to the parent Fc domain of human IgG1, a “varianthuman IgG4 Fc domain” is compared to the parent Fc domain human IgG4,etc.

By “Fc” or “Fc region” or “Fc domain” as used herein is meant thepolypeptide comprising the CH2-CH3 domains of an IgG molecule, and insome cases, inclusive of the hinge. In EU numbering for human IgG1, theCH2-CH3 domain comprises amino acids 231 to 447, and the hinge is 216 to230. Thus the definition of “Fc domain” includes both amino acids231-447 (CH2-CH3) or 216-447 (hinge-CH2-CH3), or fragments thereof. An“Fc fragment” in this context may contain fewer amino acids from eitheror both of the N- and C-termini but still retains the ability to form adimer with another Fc domain or Fc fragment as can be detected usingstandard methods, generally based on size (e.g. non-denaturingchromatography, size exclusion chromatography, etc.) Human IgG Fcdomains are of particular use in the present invention, and can be theFc domain from human IgG1, IgG2 or IgG4.

A “variant Fc domain” contains amino acid modifications as compared to aparental Fc domain. Thus, a “variant human IgG1 Fc domain” is one thatcontains amino acid modifications (generally amino acid substitutions,although in the case of ablation variants, amino acid deletions areincluded) as compared to the human IgG1 Fc domain. In general, variantFc domains have at least about 80, 85, 90, 95, 97, 98 or 99 percentidentity to the corresponding parental human IgG Fc domain (using theidentity algorithms discussed below, with one embodiment utilizing theBLAST algorithm as is known in the art, using default parameters).Alternatively, the variant Fc domains can have from 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 11, 12, 13, 14, 15, 16, 17, 18, 19 or20 amino acid modifications as compared to the parental Fc domain.Alternatively, the variant Fc domains can have up to 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 11, 12, 13, 14, 15, 16, 17, 18, 19or 20 amino acid modifications as compared to the parental Fc domain.Additionally, as discussed herein, the variant Fc domains herein stillretain the ability to form a dimer with another Fc domain as measuredusing known techniques as described herein, such as non-denaturing gelelectrophoresis.

By “heavy chain constant region” herein is meant the CH1-hinge-CH2-CH3portion of an antibody (or fragments thereof), excluding the variableheavy domain; in EU numbering of human IgG1 this is amino acids 118-447By “heavy chain constant region fragment” herein is meant a heavy chainconstant region that contains fewer amino acids from either or both ofthe N- and C-termini but still retains the ability to form a dimer withanother heavy chain constant region.

By “position” as used herein is meant a location in the sequence of aprotein. Positions may be numbered sequentially, or according to anestablished format, for example the EU index for antibody numbering.

By “target antigen” as used herein is meant the molecule that is boundspecifically by the antigen binding domain comprising the variableregions of a given antibody. As discussed below, in the present case thetarget antigens are checkpoint inhibitor proteins.

By “strandedness” in the context of the monomers of the heterodimericantibodies of the invention herein is meant that, similar to the twostrands of DNA that “match”, heterodimerization variants areincorporated into each monomer so as to preserve the ability to “match”to form heterodimers. For example, if some pI variants are engineeredinto monomer A (e.g. making the pI higher) then steric variants that are“charge pairs” that can be utilized as well do not interfere with the pIvariants, e.g. the charge variants that make a pI higher are put on thesame “strand” or “monomer” to preserve both functionalities. Similarly,for “skew” variants that come in pairs of a set as more fully outlinedbelow, the skilled artisan will consider pI in deciding into whichstrand or monomer one set of the pair will go, such that pI separationis maximized using the pI of the skews as well.

By “target cell” as used herein is meant a cell that expresses a targetantigen.

By “host cell” in the context of producing a bispecific antibodyaccording to the invention herein is meant a cell that contains theexogeneous nucleic acids encoding the components of the bispecificantibody and is capable of expressing the bispecific antibody undersuitable conditions. Suitable host cells are discussed below.

By “variable region” or “variable domain” as used herein is meant theregion of an immunoglobulin that comprises one or more Ig domainssubstantially encoded by any of the VK, VX, and/or VH genes that make upthe kappa, lambda, and heavy chain immunoglobulin genetic locirespectively, and contains the CDRs that confer antigen specificity.Thus, a “variable heavy domain” pairs with a “variable light domain” toform an antigen binding domain (“ABD”). In addition, each variabledomain comprises three hypervariable regions (“complementary determiningregions,” “CDRs”) (vhCDR1, vhCDR2 and vhCDR3 for the variable heavydomain and vlCDR1, vlCDR2 and vlCDR3 for the variable light domain) andfour framework (FR) regions, arranged from amino-terminus tocarboxy-terminus in the following order: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

By “wild type or WT” herein is meant an amino acid sequence or anucleotide sequence that is found in nature, including allelicvariations. A WT protein has an amino acid sequence or a nucleotidesequence that has not been intentionally modified.

The invention provides a number of antibody domains that have sequenceidentity to human antibody domains. Sequence identity between twosimilar sequences (e.g., antibody variable domains) can be measured byalgorithms such as that of Smith, T. F. & Waterman, M. S. (1981)“Comparison Of Biosequences,” Adv. Appl. Math. 2:482 [local homologyalgorithm]; Needleman, S. B. & Wunsch, CD. (1970) “A General MethodApplicable To The Search For Similarities In The Amino Acid Sequence OfTwo Proteins,” J. Mol. Biol.48:443 [homology alignment algorithm],Pearson, W. R. & Lipman, D. J. (1988) “Improved Tools For BiologicalSequence Comparison,” Proc. Natl. Acad. Sci. (U.S.A.) 85:2444 [searchfor similarity method]; or Altschul, S. F. et al, (1990) “Basic LocalAlignment Search Tool,” J. Mol. Biol. 215:403-10, the “BLAST” algorithm,see https://blast.ncbi.nlm.nih.gov/Blast.cgi. When using any of theaforementioned algorithms, the default parameters (for Window length,gap penalty, etc) are used. In one embodiment, sequence identity is doneusing the BLAST algorithm, using default parameters

The antibodies of the present invention are generally isolated orrecombinant. “Isolated,” when used to describe the various polypeptidesdisclosed herein, means a polypeptide that has been identified andseparated and/or recovered from a cell or cell culture from which it wasexpressed. Ordinarily, an isolated polypeptide will be prepared by atleast one purification step. An “isolated antibody,” refers to anantibody which is substantially free of other antibodies havingdifferent antigenic specificities. “Recombinant” means the antibodiesare generated using recombinant nucleic acid techniques in exogeneoushost cells, and they can be isolated as well.

“Specific binding” or “specifically binds to” or is “specific for” aparticular antigen or an epitope means binding that is measurablydifferent from a non-specific interaction. Specific binding can bemeasured, for example, by determining binding of a molecule compared tobinding of a control molecule, which generally is a molecule of similarstructure that does not have binding activity. For example, specificbinding can be determined by competition with a control molecule that issimilar to the target.

Specific binding for a particular antigen or an epitope can beexhibited, for example, by an antibody having a KD for an antigen orepitope of at least about 10⁻⁴ M, at least about 10⁻⁵ M, at least about10⁻⁶ M, at least about 10⁻⁷ M, at least about 10⁻⁸ M, at least about10⁻⁹ M, alternatively at least about 10⁻¹⁰ M, at least about 10⁻¹¹ M, atleast about 10⁻¹²M, or greater, where KD refers to a dissociation rateof a particular antibody-antigen interaction. Typically, an antibodythat specifically binds an antigen will have a KD that is 20-, 50-,100-, 500-, 1000-, 5,000-, 10,000- or more times greater for a controlmolecule relative to the antigen or epitope.

Also, specific binding for a particular antigen or an epitope can beexhibited, for example, by an antibody having a KA or Ka for an antigenor epitope of at least 20-, 50-, 100-, 500-, 1000-, 5,000-, 10,000- ormore times greater for the epitope relative to a control, where KA or Karefers to an association rate of a particular antibody-antigeninteraction. Binding affinity is generally measured using a Biacore, SPRor BLI assay.

E. Antibodies

In one aspect, provided herein are compositions that bind to SSTR2(e.g., anti-SSTR2 antibodies). In certain embodiments, the antibodybinds to human SSTR2 (FIG. 11). Subject anti-SSTR2 antibodies includemonospecific SSTR2 antibodies, as well as multi-specific (e.g.,bispecific) anti-SSTR2 antibodies. In certain embodiments, theanti-SSTR2 antibody has a format according to any one of the antibodyformats depicted in FIG. 1.

In some embodiments, the subject compositions include an SSTR2 bindingdomain. In some embodiments, the composition includes an antibody havingan SSTR2 binding domain. Antibodies provided herein include one, two,three, four, and five or more SSTR2 binding domains. In certainembodiments, the SSTR2 binding domain includes the vhCDR1, vhCDR2,vhCDR3, vlCDR1, vlCDR2 and vlCDR3 sequences of an SSTR2 binding domainselected from the group consisting of those depicted in FIG. 11. In someembodiments, the SSTR2 binding domain includes the underlined vhCDR1,vhCDR2, vhCDR3, vlCDR1, vlCDR2 and vlCDR3 sequences of an SSTR2 bindingdomain selected from those depicted in FIG. 11. In some embodiments, theSSTR2 binding domain includes the variable heavy domain and variablelight domain of an SSTR2 binding domain selected from those depicted inFIG. 11. SSTR2 binding domains depicted in FIG. 11 include anti-SSTR2H1.143_L1.30; anti-SSTR2 H1 L1.1; anti-SSTR2 H1.107_L1.30; anti-SSTR2H1.107 L1.67; anti-SSTR2 H1.107 L1.108; anti-SSTR2 H1.107 L1.111;anti-SSTR2 H1.107_L1.114; anti-SSTR2 H1.107_L1.102; anti-SSTR2H1.107_L1.110; anti-SSTR2 H1.125 L1.30; anti-SSTR2 H1.125 L1.67;anti-SSTR2 H1.125 L1.108; anti-SSTR2 H1.125_L1.111; anti-SSTR2H1.125_L1.114; Anti-SSTR2 H1.125_L1.102; and anti-SSTR2 H1.125_L1.10. Inan exemplary embodiment, the antibody includes an anti-SSTR2H1.143_L1.30 binding domain.

In some embodiments, the antibody is a bispecific antibody that bindsSSTR2 and CD3. Such antibodies include a CD3 binding domain and at leastone SSTR2 binding domain. Any suitable SSTR2 binding domain can beincluded in the anti-SSTR2×anti-CD3 bispecific antibody. In someembodiments, the anti-SSTR2×anti-CD3 bispecific antibody includes one,two, three, four or more SSTR2 binding domains, including but notlimited to those depicted in FIG. 11. In certain embodiments, theanti-SSTR2×anti-CD3 antibody includes a SSTR2 binding domain thatincludes the vhCDR1, vhCDR2, vhCDR3, vlCDR1, vlCDR2 and vlCDR3 sequencesof an SSTR2 binding domain selected from the group consisting of thosedepicted in Figures FIG. 11. In some embodiments, theanti-SSTR2×anti-CD3 antibody includes a SSTR2 binding domain thatincludes the underlined vhCDR1, vhCDR2, vhCDR3, vlCDR1, vlCDR2 andvlCDR3 sequences of an SSTR2 binding domain selected from the groupconsisting of those depicted in FIG. 11. In some embodiments, theanti-SSTR2×anti-CD3 antibody includes a SSTR2 binding domain thatincludes the variable heavy domain and variable light domain of an SSTR2binding domain selected from the group consisting of those depicted inFIG. 11. In an exemplary embodiment, the anti-SSTR2×anti-CD3 antibodyincludes an anti-SSTR2 H1.143_L1.30 binding domain.

The anti-SSTR2×anti-CD3 antibody provided herein can include anysuitable CD3 binding domain. In certain embodiments, theanti-SSTR2×anti-CD3 antibody includes a CD3 binding domain that includesthe vhCDR1, vhCDR2, vhCDR3, vlCDR1, vlCDR2 and vlCDR3 sequences of a CD3binding domain selected from the group consisting of those depicted inFIGS. 12 and 13. In some embodiments, the anti-SSTR2×anti-CD3 antibodyincludes a CD3 binding domain that includes the underlined vhCDR1,vhCDR2, vhCDR3, vlCDR1, vlCDR2 and vlCDR3 sequences of a CD3 bindingdomain selected from the group consisting of those depicted in FIG. 12or 13. In some embodiments, the anti-SSTR2×anti-CD3 antibody includes aCD3 binding domain that includes the variable heavy domain and variablelight domain of a CD3 binding domain selected from the group consistingof those depicted in FIG. 12 or 13. In some embodiments, the CD3 bindingdomain is selected from anti-CD3 H1.30_L1.47, anti-CD3 H1.32_L1.47;anti-CD3 H1.89 L1.48; anti-CD3 H1.90 L1.47; Anti-CD3 H1.33 L1.47; andanti-CD3 H1.31 L1.47.

As used herein, term “antibody” is used generally. Antibodies that finduse in the present invention can take on a number of formats asdescribed herein, including traditional antibodies as well as antibodyderivatives, fragments and mimetics, described herein.

Traditional antibody structural units typically comprise a tetramer.Each tetramer is typically composed of two identical pairs ofpolypeptide chains, each pair having one “light” (typically having amolecular weight of about 25 kDa) and one “heavy” chain (typicallyhaving a molecular weight of about 50-70 kDa). Human light chains areclassified as kappa and lambda light chains. The present invention isdirected to the IgG class, which has several subclasses, including, butnot limited to IgG1, IgG2, IgG3, and IgG4. It should be noted that IgG1has different allotypes with polymorphisms at 356 (D or E) and 358 (L orM). The sequences depicted herein use the 356D/358M allotype, howeverthe other allotype is included herein. That is, any sequence inclusiveof an IgG1 Fc domain included herein can have 356E/358L replacing the356D/358M allotype.

In addition, many of the antibodies herein have at least one thecysteines at position 220 replaced by a serine; generally this is the onthe “scFv monomer” side for most of the sequences depicted herein,although it can also be on the “Fab monomer” side, or both, to reducedisulfide formation. Specifically included within the sequences hereinare one or both of these cysteines replaced (C220S).

Thus, “isotype” as used herein is meant any of the subclasses ofimmunoglobulins defined by the chemical and antigenic characteristics oftheir constant regions. It should be understood that therapeuticantibodies can also comprise hybrids of isotypes and/or subclasses. Forexample, as shown in US Publication 2009/0163699, incorporated byreference, the present invention includes the use of human IgG1/G2hybrids.

The hypervariable region generally encompasses amino acid residues fromabout amino acid residues 24-34 (LCDR1; “L” denotes light chain), 50-56(LCDR2) and 89-97 (LCDR3) in the light chain variable region and aroundabout 31-35B (HCDR1; “H” denotes heavy chain), 50-65 (HCDR2), and 95-102(HCDR3) in the heavy chain variable region; Kabat et al., SEQUENCES OFPROTEINS OF IMMUNOLOGICAL INTEREST, 5th Ed. Public Health Service,National Institutes of Health, Bethesda, Md. (1991) and/or thoseresidues forming a hypervariable loop (e.g. residues 26-32 (LCDR1),50-52 (LCDR2) and 91-96 (LCDR3) in the light chain variable region and26-32 (HCDR1), 53-55 (HCDR2) and 96-101 (HCDR3) in the heavy chainvariable region; Chothia and Lesk (1987) J. Mol. Biol. 196:901-917.Specific CDRs of the invention are described below.

As will be appreciated by those in the art, the exact numbering andplacement of the CDRs can be different among different numberingsystems. However, it should be understood that the disclosure of avariable heavy and/or variable light sequence includes the disclosure ofthe associated (inherent) CDRs. Accordingly, the disclosure of eachvariable heavy region is a disclosure of the vhCDRs (e.g. vhCDR1, vhCDR2and vhCDR3) and the disclosure of each variable light region is adisclosure of the vlCDRs (e.g. vlCDR1, vlCDR2 and vlCDR3). A usefulcomparison of CDR numbering is as below, see Lafranc et al., Dev. Comp.Immunol. 27(1):55-77 (2003):

TABLE 1 Kabat + Chothia IMGT Kabat AbM Chothia Contact Xencor vhCDR126-35 27-38 31-35 26-35 26-32 30-35 27-35 vhCDR2 50-65 56-65 50-65 50-5852-56 47-58 54-61 vhCDR3  95-102 105-117  95-102  95-102  95-102  93-101103-116 vlCDR1 24-34 27-38 24-34 24-34 24-34 30-36 27-38 vlCDR2 50-5656-65 50-56 50-56 50-56 46-55 56-62 vlCDR3 89-97 105-117 89-97 89-9789-97 89-96  97-105

Throughout the present specification, the Kabat numbering system isgenerally used when referring to a residue in the variable domain(approximately, residues 1-107 of the light chain variable region andresidues 1-113 of the heavy chain variable region) and the EU numberingsystem for Fc regions (e.g, Kabat et al., supra (1991)).

Another type of Ig domain of the heavy chain is the hinge region. By“hinge” or “hinge region” or “antibody hinge region” or “hinge domain”herein is meant the flexible polypeptide comprising the amino acidsbetween the first and second constant domains of an antibody.Structurally, the IgG CH1 domain ends at EU position 215, and the IgGCH2 domain begins at residue EU position 231. Thus for IgG the antibodyhinge is herein defined to include positions 216 (E216 in IgG1) to 230(p230 in IgG1), wherein the numbering is according to the EU index as inKabat. In some cases, a “hinge fragment” is used, which contains feweramino acids at either or both of the N- and C-termini of the hingedomain. As noted herein, pI variants can be made in the hinge region aswell.

The light chain generally comprises two domains, the variable lightdomain (containing the light chain CDRs and together with the variableheavy domains forming the Fv region), and a constant light chain region(often referred to as CL or CK).

Another region of interest for additional substitutions, outlined below,is the Fc region.

The present invention provides a large number of different CDR sets. Inthis case, a “full CDR set” comprises the three variable light and threevariable heavy CDRs, e.g. a vlCDR1, vlCDR2, vlCDR3, vhCDR1, vhCDR2 andvhCDR3. These can be part of a larger variable light or variable heavydomain, respectfully. In addition, as more fully outlined herein, thevariable heavy and variable light domains can be on separate polypeptidechains, when a heavy and light chain is used (for example when Fabs areused), or on a single polypeptide chain in the case of scFv sequences.

The CDRs contribute to the formation of the antigen-binding, or morespecifically, epitope binding site of antibodies. “Epitope” refers to adeterminant that interacts with a specific antigen binding site in thevariable region of an antibody molecule known as a paratope. Epitopesare groupings of molecules such as amino acids or sugar side chains andusually have specific structural characteristics, as well as specificcharge characteristics. A single antigen may have more than one epitope.

The epitope may comprise amino acid residues directly involved in thebinding (also called immunodominant component of the epitope) and otheramino acid residues, which are not directly involved in the binding,such as amino acid residues which are effectively blocked by thespecifically antigen binding peptide; in other words, the amino acidresidue is within the footprint of the specifically antigen bindingpeptide.

Epitopes may be either conformational or linear. A conformationalepitope is produced by spatially juxtaposed amino acids from differentsegments of the linear polypeptide chain. A linear epitope is oneproduced by adjacent amino acid residues in a polypeptide chain.Conformational and nonconformational epitopes may be distinguished inthat the binding to the former but not the latter is lost in thepresence of denaturing solvents.

An epitope typically includes at least 3, and more usually, at least 5or 8-10 amino acids in a unique spatial conformation. Antibodies thatrecognize the same epitope can be verified in a simple immunoassayshowing the ability of one antibody to block the binding of anotherantibody to a target antigen, for example “binning.” As outlined below,the invention not only includes the enumerated antigen binding domainsand antibodies herein, but those that compete for binding with theepitopes bound by the enumerated antigen binding domains.

Thus, the present invention provides different antibody domains. Asdescribed herein and known in the art, the heterodimeric antibodies ofthe invention comprise different domains within the heavy and lightchains, which can be overlapping as well. These domains include, but arenot limited to, the Fc domain, the CH1 domain, the CH2 domain, the CH3domain, the hinge domain, the heavy constant domain (CH1-hinge-Fc domainor CH1-hinge-CH2-CH3), the variable heavy domain, the variable lightdomain, the light constant domain, Fab domains and scFv domains.

Thus, the “Fc domain” includes the —CH2-CH3 domain, and optionally ahinge domain (—H-CH2-CH3). In the embodiments herein, when a scFv isattached to an Fc domain, it is the C-terminus of the scFv constructthat is attached to all or part of the hinge of the Fc domain; forexample, it is generally attached to the sequence EPKS which is thebeginning of the hinge. The heavy chain comprises a variable heavydomain and a constant domain, which includes a CH1-optional hinge-Fcdomain comprising a CH2-CH3. The light chain comprises a variable lightchain and the light constant domain. A scFv comprises a variable heavychain, an scFv linker, and a variable light domain. In most of theconstructs and sequences outlined herein, the C-terminus of the variableheavy chain is attached to the N-terminus of the scFv linker, theC-terminus of which is attached to the N-terminus of a variable lightchain (N-vh-linker-vl-C) although that can be switched(N-vl-linker-vh-C).

Some embodiments of the invention comprise at least one scFv domain,which, while not naturally occurring, generally includes a variableheavy domain and a variable light domain, linked together by a scFvlinker. As outlined herein, while the scFv domain is generally from N-to C-terminus oriented as vh-scFv linker-vl, this can be reversed forany of the scFv domains (or those constructed using vh and vl sequencesfrom Fabs), to vl-scFv linker-vh, with optional linkers at one or bothends depending on the format (see generally FIG. 1).

As shown herein, there are a number of suitable linkers (for use aseither domain linkers or scFv linkers) that can be used to covalentlyattach the recited domains, including traditional peptide bonds,generated by recombinant techniques. In some embodiments, the linkerpeptide may predominantly include the following amino acid residues:Gly, Ser, Ala, or Thr. The linker peptide should have a length that isadequate to link two molecules in such a way that they assume thecorrect conformation relative to one another so that they retain thedesired activity. In one embodiment, the linker is from about 1 to 50amino acids in length, preferably about 1 to 30 amino acids in length.In one embodiment, linkers of 1 to 20 amino acids in length may be used,with from about 5 to about 10 amino acids finding use in someembodiments. Useful linkers include glycine-serine polymers, includingfor example (GS)n, (GSGGS)n, (GGGGS)n, and (GGGS)n, where n is aninteger of at least one (and generally from 3 to 4), glycine-alaninepolymers, alanine-serine polymers, and other flexible linkers.Alternatively, a variety of nonproteinaceous polymers, including but notlimited to polyethylene glycol (PEG), polypropylene glycol,polyoxyalkylenes, or copolymers of polyethylene glycol and polypropyleneglycol, may find use as linkers.

Other linker sequences may include any sequence of any length of CL/CH1domain but not all residues of CL/CH1 domain; for example the first 5-12amino acid residues of the CL/CH1 domains. Linkers can be derived fromimmunoglobulin light chain, for example Cκ or Cλ. Linkers can be derivedfrom immunoglobulin heavy chains of any isotype, including for exampleCγ1, Cγ2, Cγ3, Cγ4, Cα1, Cα2, Cδ, Cε, and Cμ. Linker sequences may alsobe derived from other proteins such as Ig-like proteins (e.g. TCR, FcR,KIR), hinge region-derived sequences, and other natural sequences fromother proteins.

In some embodiments, the linker is a “domain linker”, used to link anytwo domains as outlined herein together. For example, in FIG. 1F, theremay be a domain linker that attaches the C-terminus of the CH1 domain ofthe Fab to the N-terminus of the scFv, with another optional domainlinker attaching the C-terminus of the scFv to the CH2 domain (althoughin many embodiments the hinge is used as this domain linker). While anysuitable linker can be used, many embodiments utilize a glycine-serinepolymer as the domain linker, including for example (GS)n, (GSGGS)n,(GGGGS)n, and (GGGS)n, where n is an integer of at least one (andgenerally from 3 to 4 to 5) as well as any peptide sequence that allowsfor recombinant attachment of the two domains with sufficient length andflexibility to allow each domain to retain its biological function. Insome cases, and with attention being paid to “strandedness”, as outlinedbelow, charged domain linkers, as used in some embodiments of scFvlinkers can be used.

In some embodiments, the linker is a scFv linker, used to covalentlyattach the vh and vl domains as discussed herein. In many cases, thescFv linker is a charged scFv linker, a number of which are shown inFIG. 7. Accordingly, the present invention further provides charged scFvlinkers, to facilitate the separation in pI between a first and a secondmonomer. That is, by incorporating a charged scFv linker, eitherpositive or negative (or both, in the case of scaffolds that use scFvson different monomers), this allows the monomer comprising the chargedlinker to alter the pI without making further changes in the Fc domains.These charged linkers can be substituted into any scFv containingstandard linkers. Again, as will be appreciated by those in the art,charged scFv linkers are used on the correct “strand” or monomer,according to the desired changes in pI. For example, as discussedherein, to make triple F format heterodimeric antibody, the original pIof the Fv region for each of the desired antigen binding domains arecalculated, and one is chosen to make an scFv, and depending on the pI,either positive or negative linkers are chosen.

Charged domain linkers can also be used to increase the pI separation ofthe monomers of the invention as well, and thus those included in FIG. 7can be used in any embodiment herein where a linker is utilized.

In particular, the formats depicted in FIG. 1 are antibodies, usuallyreferred to as “heterodimeric antibodies”, meaning that the protein hasat least two associated Fc sequences self-assembled into a heterodimericFc domain and at least two Fv regions, whether as Fabs or as scFvs.

F. Chimeric and Humanized Antibodies

In certain embodiments, the antibodies of the invention comprise a heavychain variable region from a particular germline heavy chainimmunoglobulin gene and/or a light chain variable region from aparticular germline light chain immunoglobulin gene. For example, suchantibodies may comprise or consist of a human antibody comprising heavyor light chain variable regions that are “the product of” or “derivedfrom” a particular germline sequence. A human antibody that is “theproduct of” or “derived from” a human germline immunoglobulin sequencecan be identified as such by comparing the amino acid sequence of thehuman antibody to the amino acid sequences of human germlineimmunoglobulins and selecting the human germline immunoglobulin sequencethat is closest in sequence (i.e., greatest % identity) to the sequenceof the human antibody (using the methods outlined herein). A humanantibody that is “the product of” or “derived from” a particular humangermline immunoglobulin sequence may contain amino acid differences ascompared to the germline sequence, due to, for example,naturally-occurring somatic mutations or intentional introduction ofsite-directed mutation. However, a humanized antibody typically is atleast 90% identical in amino acids sequence to an amino acid sequenceencoded by a human germline immunoglobulin gene and contains amino acidresidues that identify the antibody as being derived from humansequences when compared to the germline immunoglobulin amino acidsequences of other species (e.g., murine germline sequences). In certaincases, a humanized antibody may be at least 95, 96, 97, 98 or 99%, oreven at least 96%, 97%, 98%, or 99% identical in amino acid sequence tothe amino acid sequence encoded by the germline immunoglobulin gene.Typically, a humanized antibody derived from a particular human germlinesequence will display no more than 10-20 amino acid differences from theamino acid sequence encoded by the human germline immunoglobulin gene(prior to the introduction of any skew, pI and ablation variants herein;that is, the number of variants is generally low, prior to theintroduction of the variants of the invention). In certain cases, thehumanized antibody may display no more than 5, or even no more than 4,3, 2, or 1 amino acid difference from the amino acid sequence encoded bythe germline immunoglobulin gene (again, prior to the introduction ofany skew, pI and ablation variants herein; that is, the number ofvariants is generally low, prior to the introduction of the variants ofthe invention).

In one embodiment, the parent antibody has been affinity matured, as isknown in the art. Structure-based methods may be employed forhumanization and affinity maturation, for example as described in U.S.Ser. No. 11/004,590. Selection based methods may be employed to humanizeand/or affinity mature antibody variable regions, including but notlimited to methods described in Wu et al., 1999, J. Mol. Biol.294:151-162; Baca et al., 1997, J. Biol. Chem. 272(16):10678-10684;Rosok et al., 1996, J. Biol. Chem. 271(37): 22611-22618; Rader et al.,1998, Proc. Natl. Acad. Sci. USA 95: 8910-8915; Krauss et al., 2003,Protein Engineering 16(10):753-759, all entirely incorporated byreference. Other humanization methods may involve the grafting of onlyparts of the CDRs, including but not limited to methods described inU.S. Ser. No. 09/810,510; Tan et al., 2002, J. Immunol. 169:1119-1125;De Pascalis et al., 2002, J. Immunol. 169:3076-3084, all entirelyincorporated by reference.

G. Heterodimeric Antibodies

Accordingly, in some embodiments, the subject antibody is aheterodimeric antibody that relies on the use of two different heavychain variant Fc sequences. Such an antibody will self-assemble to forma heterodimeric Fc domain and heterodimeric antibody.

The present invention is directed to novel constructs to provideheterodimeric antibodies that allow binding to more than one antigen orligand, e.g. to allow for bispecific binding (e.g., anti-SSTR2 andanti-CD3 binding). The heterodimeric antibody constructs are based onthe self-assembling nature of the two Fc domains of the heavy chains ofantibodies, e.g. two “monomers” that assemble into a “dimer”.Heterodimeric antibodies are made by altering the amino acid sequence ofeach monomer as more fully discussed below. Thus, the present inventionis generally directed to the creation of heterodimeric antibodies whichcan co-engage antigens (e.g., SSTR2 and CD3) in several ways, relying onamino acid variants in the constant regions that are different on eachchain to promote heterodimeric formation and/or allow for ease ofpurification of heterodimers over the homodimers.

Thus, the present invention provides bispecific antibodies. In someembodiments, the present invention provides bispecific antibodies thatinclude an SSTR2 binding domain. In some embodiments, the bispecificantibody is an anti-SSTR2×anti-CD3 bispecific antibody. An ongoingproblem in antibody technologies is the desire for “bispecific”antibodies that bind to two different antigens simultaneously, ingeneral thus allowing the different antigens to be brought intoproximity and resulting in new functionalities and new therapies. Ingeneral, these antibodies are made by including genes for each heavy andlight chain into the host cells. This generally results in the formationof the desired heterodimer (A-B), as well as the two homodimers (A-A andB-B (not including the light chain heterodimeric issues)). However, amajor obstacle in the formation of bispecific antibodies is thedifficulty in purifying the heterodimeric antibodies away from thehomodimeric antibodies and/or biasing the formation of the heterodimerover the formation of the homodimers.

There are a number of mechanisms that can be used to generate theheterodimers of the present invention. In addition, as will beappreciated by those in the art, these mechanisms can be combined toensure high heterodimerization. Thus, amino acid variants that lead tothe production of heterodimers are referred to as “heterodimerizationvariants”. As discussed below, heterodimerization variants can includesteric variants (e.g. the “knobs and holes” or “skew” variants describedbelow and the “charge pairs” variants described below) as well as “pIvariants”, which allows purification of homodimers away fromheterodimers. As is generally described in WO2014/145806, herebyincorporated by reference in its entirety and specifically as below forthe discussion of “heterodimerization variants”, useful mechanisms forheterodimerization include “knobs and holes” (“KIH”; sometimes herein as“skew” variants (see discussion in WO2014/145806), “electrostaticsteering” or “charge pairs” as described in WO2014/145806, pI variantsas described in WO2014/145806, and general additional Fc variants asoutlined in WO2014/145806 and below.

In the present invention, there are several basic mechanisms that canlead to ease of purifying heterodimeric antibodies; one relies on theuse of pI variants, such that each monomer has a different pI, thusallowing the isoelectric purification of A-A, A-B and B-B dimericproteins. Alternatively, some scaffold formats, such as the “triple F”format, also allows separation on the basis of size. As is furtheroutlined below, it is also possible to “skew” the formation ofheterodimers over homodimers. Thus, a combination of stericheterodimerization variants and pI or charge pair variants findparticular use in the invention.

In general, embodiments of particular use in the present invention relyon sets of variants that include skew variants, which encourageheterodimerization formation over homodimerization formation, coupledwith pI variants, which increase the pI difference between the twomonomers to facilitate purification of heterodimers away fromhomodimers.

Additionally, as more fully outlined below, depending on the format ofthe heterodimer antibody, pI variants can be either contained within theconstant and/or Fc domains of a monomer, or charged linkers, eitherdomain linkers or scFv linkers, can be used. That is, scaffolds thatutilize scFv(s) such as the Triple F, or “bottle opener”, format caninclude charged scFv linkers (either positive or negative), that give afurther pI boost for purification purposes. As will be appreciated bythose in the art, some Triple F formats are useful with just chargedscFv linkers and no additional pI adjustments, although the inventiondoes provide pI variants that are on one or both of the monomers, and/orcharged domain linkers as well. In addition, additional amino acidengineering for alternative functionalities may also confer pI changes,such as Fc, FcRn and KO variants.

In the present invention that utilizes pI as a separation mechanism toallow the purification of heterodimeric proteins, amino acid variantscan be introduced into one or both of the monomer polypeptides; that is,the pI of one of the monomers (referred to herein for simplicity as“monomer A”) can be engineered away from monomer B, or both monomer Aand B change be changed, with the pI of monomer A increasing and the pIof monomer B decreasing. As is outlined more fully below, the pI changesof either or both monomers can be done by removing or adding a chargedresidue (e.g. a neutral amino acid is replaced by a positively ornegatively charged amino acid residue, e.g. glycine to glutamic acid),changing a charged residue from positive or negative to the oppositecharge (aspartic acid to lysine) or changing a charged residue to aneutral residue (e.g. loss of a charge; lysine to serine.). A number ofthese variants are shown in the Figures.

Accordingly, this embodiment of the present invention provides forcreating a sufficient change in pI in at least one of the monomers suchthat heterodimers can be separated from homodimers. As will beappreciated by those in the art, and as discussed further below, thiscan be done by using a “wild type” heavy chain constant region and avariant region that has been engineered to either increase or decreaseit's pI (wt A−+B or wt A−−B), or by increasing one region and decreasingthe other region (A+−B− or A−B+).

Thus, in general, a component of some embodiments of the presentinvention are amino acid variants in the constant regions of antibodiesthat are directed to altering the isoelectric point (pI) of at leastone, if not both, of the monomers of a dimeric protein to form “pIantibodies”) by incorporating amino acid substitutions (“pI variants” or“pI substitutions”) into one or both of the monomers. As shown herein,the separation of the heterodimers from the two homodimers can beaccomplished if the pIs of the two monomers differ by as little as 0.1pH unit, with 0.2, 0.3, 0.4 and 0.5 or greater all finding use in thepresent invention.

As will be appreciated by those in the art, the number of pI variants tobe included on each or both monomer(s) to get good separation willdepend in part on the starting pI of the components, for example in thetriple F format, the starting pI of the scFv and Fab of interest. Thatis, to determine which monomer to engineer or in which “direction” (e.g.more positive or more negative), the Fv sequences of the two targetantigens are calculated and a decision is made from there. As is knownin the art, different Fvs will have different starting pIs which areexploited in the present invention. In general, as outlined herein, thepIs are engineered to result in a total pI difference of each monomer ofat least about 0.1 logs, with 0.2 to 0.5 being preferred as outlinedherein.

Furthermore, as will be appreciated by those in the art and outlinedherein, in some embodiments, heterodimers can be separated fromhomodimers on the basis of size. As shown in FIG. 1, for example,several of the formats allow separation of heterodimers and homodimerson the basis of size.

In the case where pI variants are used to achieve heterodimerization, byusing the constant region(s) of the heavy chain(s), a more modularapproach to designing and purifying bispecific proteins, includingantibodies, is provided. Thus, in some embodiments, heterodimerizationvariants (including skew and purification heterodimerization variants)are not included in the variable regions, such that each individualantibody must be engineered. In addition, in some embodiments, thepossibility of immunogenicity resulting from the pI variants issignificantly reduced by importing pI variants from different IgGisotypes such that pI is changed without introducing significantimmunogenicity. Thus, an additional problem to be solved is theelucidation of low pI constant domains with high human sequence content,e.g. the minimization or avoidance of non-human residues at anyparticular position.

A side benefit that can occur with this pI engineering is also theextension of serum half-life and increased FcRn binding. That is, asdescribed in U.S. Ser. No. 13/194,904 (incorporated by reference in itsentirety), lowering the pI of antibody constant domains (including thosefound in antibodies and Fc fusions) can lead to longer serum retentionin vivo. These pI variants for increased serum half life also facilitatepI changes for purification.

In addition, it should be noted that the pI variants of theheterodimerization variants give an additional benefit for the analyticsand quality control process of bispecific antibodies, as the ability toeither eliminate, minimize and distinguish when homodimers are presentis significant. Similarly, the ability to reliably test thereproducibility of the heterodimeric antibody production is important.

Heterodimerization Variants

The present invention provides heterodimeric proteins, includingheterodimeric antibodies in a variety of formats, which utilizeheterodimeric variants to allow for heterodimeric formation and/orpurification away from homodimers.

There are a number of suitable pairs of sets of heterodimerization skewvariants. These variants come in “pairs” of “sets”. That is, one set ofthe pair is incorporated into the first monomer and the other set of thepair is incorporated into the second monomer. It should be noted thatthese sets do not necessarily behave as “knobs in holes” variants, witha one-to-one correspondence between a residue on one monomer and aresidue on the other; that is, these pairs of sets form an interfacebetween the two monomers that encourages heterodimer formation anddiscourages homodimer formation, allowing the percentage of heterodimersthat spontaneously form under biological conditions to be over 90%,rather than the expected 50% (25% homodimer A/A:50% heterodimer A/B:25%homodimer B/B).

Steric Variants

In some embodiments, the formation of heterodimers can be facilitated bythe addition of steric variants. That is, by changing amino acids ineach heavy chain, different heavy chains are more likely to associate toform the heterodimeric structure than to form homodimers with the sameFc amino acid sequences. Suitable steric variants are included in FIG.12.

One mechanism is generally referred to in the art as “knobs and holes”,referring to amino acid engineering that creates steric influences tofavor heterodimeric formation and disfavor homodimeric formation canalso optionally be used; this is sometimes referred to as “knobs andholes”, as described in U.S. Ser. No. 61/596,846, Ridgway et al.,Protein Engineering 9(7):617 (1996); Atwell et al., J. Mol. Biol. 1997270:26; U.S. Pat. No. 8,216,805, all of which are hereby incorporated byreference in their entirety. The Figures identify a number of “monomerA-monomer B” pairs that rely on “knobs and holes”. In addition, asdescribed in Merchant et al., Nature Biotech. 16:677 (1998), these“knobs and hole” mutations can be combined with disulfide bonds to skewformation to heterodimerization.

An additional mechanism that finds use in the generation of heterodimersis sometimes referred to as “electrostatic steering” as described inGunasekaran et al., J. Biol. Chem. 285(25):19637 (2010), herebyincorporated by reference in its entirety. This is sometimes referred toherein as “charge pairs”. In this embodiment, electrostatics are used toskew the formation towards heterodimerization. As those in the art willappreciate, these may also have have an effect on pI, and thus onpurification, and thus could in some cases also be considered pIvariants. However, as these were generated to force heterodimerizationand were not used as purification tools, they are classified as “stericvariants”. These include, but are not limited to, D221E/P228E/L368Epaired with D221R/P228R/K409R (e.g. these are “monomer correspondingsets) and C220E/P228E/368E paired with C220R/E224R/P228R/K409R.

Additional monomer A and monomer B variants that can be combined withother variants, optionally and independently in any amount, such as pIvariants outlined herein or other steric variants that are shown in FIG.37 of US 2012/0149876, the figure and legend and SEQ ID NOs of which areincorporated expressly by reference herein.

In some embodiments, the steric variants outlined herein can beoptionally and independently incorporated with any pI variant (or othervariants such as Fc variants, FcRn variants, etc.) into one or bothmonomers, and can be independently and optionally included or excludedfrom the proteins of the invention.

A list of suitable skew variants is found in FIG. 3, with FIG. 8 showingsome pairs of particular utility in many embodiments. Of particular usein many embodiments are the pairs of sets including, but not limited to,S364K/E357Q:L368D/K370S; L368D/K370S:S364K; L368E/K370S:S364K;T411T/E360E/Q362E:D401K; L368D/K370S:S364K/E357L and K370S:S364K/E357Q.In terms of nomenclature, the pair “S364K/E357Q:L368D/K370S” means thatone of the monomers has the double variant set S364K/E357Q and the otherhas the double variant set L368D/K370S.

pI (Isoelectric Point) Variants for Heterodimers

In general, as will be appreciated by those in the art, there are twogeneral categories of pI variants: those that increase the pI of theprotein (basic changes) and those that decrease the pI of the protein(acidic changes). As described herein, all combinations of thesevariants can be done: one monomer may be wild type, or a variant thatdoes not display a significantly different pI from wild-type, and theother can be either more basic or more acidic. Alternatively, eachmonomer is changed, one to more basic and one to more acidic.

Preferred combinations of pI variants are shown in FIG. 4. As outlinedherein and shown in the figures, these changes are shown relative toIgG1, but all isotypes can be altered this way, as well as isotypehybrids. In the case where the heavy chain constant domain is fromIgG2-4, R133E and R133Q can also be used.

In one embodiment, for example in the FIGS. 1A, E, F, G, H and Iformats, a preferred combination of pI variants has one monomer (thenegative Fab side) comprising 208D/295E/384D/418E/421D variants(N208D/Q295E/N384D/Q418E/N421D when relative to human IgG1) and a secondmonomer (the positive scFv side) comprising a positively charged scFvlinker, including (GKPGS)4 (SEQ ID NO: 818). However, as will beappreciated by those in the art, the first monomer includes a CH1domain, including position 208. Accordingly, in constructs that do notinclude a CH1 domain (for example for antibodies that do not utilize aCH1 domain on one of the domains, for example in a dual scFv format or a“one armed” format such as those depicted in FIG. 1B, C or D), apreferred negative pI variant Fc set includes 295E/384D/418E/421Dvariants (Q295E/N384D/Q418E/N421D when relative to human IgG1).

Accordingly, in some embodiments, one monomer has a set of substitutionsfrom FIG. 4 and the other monomer has a charged linker (either in theform of a charged scFv linker because that monomer comprises an scFv ora charged domain linker, as the format dictates, which can be selectedfrom those depicted in FIG. 7).

Isotypic Variants

In addition, many embodiments of the invention rely on the “importation”of pI amino acids at particular positions from one IgG isotype intoanother, thus reducing or eliminating the possibility of unwantedimmunogenicity being introduced into the variants. A number of these areshown in FIG. 21 of US Publ. 2014/0370013, hereby incorporated byreference. That is, IgG1 is a common isotype for therapeutic antibodiesfor a variety of reasons, including high effector function. However, theheavy constant region of IgG1 has a higher pI than that of IgG2 (8.10versus 7.31). By introducing IgG2 residues at particular positions intothe IgG1 backbone, the pI of the resulting monomer is lowered (orincreased) and additionally exhibits longer serum half-life. Forexample, IgG1 has a glycine (pI 5.97) at position 137, and IgG2 has aglutamic acid (pI 3.22); importing the glutamic acid will affect the pIof the resulting protein. As is described below, a number of amino acidsubstitutions are generally required to significant affect the pI of thevariant antibody. However, it should be noted as discussed below thateven changes in IgG2 molecules allow for increased serum half-life.

In other embodiments, non-isotypic amino acid changes are made, eitherto reduce the overall charge state of the resulting protein (e.g. bychanging a higher pI amino acid to a lower pI amino acid), or to allowaccommodations in structure for stability, etc. as is more furtherdescribed below.

In addition, by pI engineering both the heavy and light constantdomains, significant changes in each monomer of the heterodimer can beseen. As discussed herein, having the pIs of the two monomers differ byat least 0.5 can allow separation by ion exchange chromatography orisoelectric focusing, or other methods sensitive to isoelectric point.

Calculating pI

The pI of each monomer can depend on the pI of the variant heavy chainconstant domain and the pI of the total monomer, including the variantheavy chain constant domain and the fusion partner. Thus, in someembodiments, the change in pI is calculated on the basis of the variantheavy chain constant domain, using the chart in the FIG. 19 of US Pub.2014/0370013. As discussed herein, which monomer to engineer isgenerally decided by the inherent pI of the Fv and scaffold regions.Alternatively, the pI of each monomer can be compared.

pI Variants that Also Confer Better FcRn In Vivo Binding

In the case where the pI variant decreases the pI of the monomer, theycan have the added benefit of improving serum retention in vivo.

Although still under examination, Fc regions are believed to have longerhalf-lives in vivo, because binding to FcRn at pH 6 in an endosomesequesters the Fc (Ghetie and Ward, 1997 Immunol Today. 18(12): 592-598,entirely incorporated by reference). The endosomal compartment thenrecycles the Fc to the cell surface. Once the compartment opens to theextracellular space, the higher pH, ˜7.4, induces the release of Fc backinto the blood. In mice, Dall′ Acqua et al. showed that Fc mutants withincreased FcRn binding at pH 6 and pH 7.4 actually had reduced serumconcentrations and the same half life as wild-type Fc (Dall′ Acqua etal. 2002, J. Immunol. 169:5171-5180, entirely incorporated byreference). The increased affinity of Fc for FcRn at pH 7.4 is thoughtto forbid the release of the Fc back into the blood. Therefore, the Fcmutations that will increase Fc's half-life in vivo will ideallyincrease FcRn binding at the lower pH while still allowing release of Fcat higher pH. The amino acid histidine changes its charge state in thepH range of 6.0 to 7.4. Therefore, it is not surprising to find Hisresidues at important positions in the Fc/FcRn complex.

Recently it has been suggested that antibodies with variable regionsthat have lower isoelectric points may also have longer serum half-lives(Igawa et al., 2010 PEDS. 23(5): 385-392, entirely incorporated byreference). However, the mechanism of this is still poorly understood.Moreover, variable regions differ from antibody to antibody. Constantregion variants with reduced pI and extended half-life would provide amore modular approach to improving the pharmacokinetic properties ofantibodies, as described herein.

Additional Fe Variants for Additional Functionality

In addition to pI amino acid variants, there are a number of useful Fcamino acid modification that can be made for a variety of reasons,including, but not limited to, altering binding to one or more FcγRreceptors, altered binding to FcRn receptors, etc.

Accordingly, the proteins of the invention can include amino acidmodifications, including the heterodimerization variants outlinedherein, which includes the pI variants and steric variants. Each set ofvariants can be independently and optionally included or excluded fromany particular heterodimeric protein.

FcγR Variants

Accordingly, there are a number of useful Fc substitutions that can bemade to alter binding to one or more of the FcγR receptors.Substitutions that result in increased binding as well as decreasedbinding can be useful. For example, it is known that increased bindingto Fc□RIIIa generally results in increased ADCC (antibody dependentcell-mediated cytotoxicity; the cell-mediated reaction whereinnonspecific cytotoxic cells that express FcγRs recognize bound antibodyon a target cell and subsequently cause lysis of the target cell).Similarly, decreased binding to FcγRIIb (an inhibitory receptor) can bebeneficial as well in some circumstances. Amino acid substitutions thatfind use in the present invention include those listed in U.S. Ser. Nos.11/124,620 (particularly FIG. 41), 11/174,287, 11/396,495, 11/538,406,all of which are expressly incorporated herein by reference in theirentirety and specifically for the variants disclosed therein. Particularvariants that find use include, but are not limited to, 236A, 239D,239E, 332E, 332D, 239D/332E, 267D, 267E, 328F, 267E/328F, 236A/332E,239D/332E/330Y, 239D, 332E/330L, 243A, 243L, 264A, 264V and 299T.

In addition, there are additional Fc substitutions that find use inincreased binding to the FcRn receptor and increased serum half life, asspecifically disclosed in U.S. Ser. No. 12/341,769, hereby incorporatedby reference in its entirety, including, but not limited to, 434S, 434A,428L, 308F, 2591, 428L/434S, 2591/308F, 4361/428L, 4361 or V/434S,436V/428L and 2591/308F/428L.

Ablation Variants

Similarly, another category of functional variants are “FcγR ablationvariants” or “Fc knock out (FcKO or KO)” variants. In these embodiments,for some therapeutic applications, it is desirable to reduce or removethe normal binding of the Fc domain to one or more or all of the Fcγreceptors (e.g. FcγR1, FcγRIIa, FcγRIIb, FcγRIIIa, etc.) to avoidadditional mechanisms of action. That is, for example, in manyembodiments, particularly in the use of bispecific antibodies that bindCD3 monovalently it is generally desirable to ablate FcγRIIIa binding toeliminate or significantly reduce ADCC activity. wherein one of the Fcdomains comprises one or more Fcγ receptor ablation variants. Theseablation variants are depicted in FIG. 14, and each can be independentlyand optionally included or excluded, with preferred aspects utilizingablation variants selected from the group consisting of G236R/L328R,E233P/L234V/L235A/G236del/S239K, E233P/L234V/L235A/G236del/S267K,E233P/L234V/L235A/G236del/S239K/A327G,E233P/L234V/L235A/G236del/S267K/A327G and E233P/L234V/L235A/G236del. Itshould be noted that the ablation variants referenced herein ablate FcγRbinding but generally not FcRn binding.

As is known in the art, the Fc domain of human IgG1 has the highestbinding to the Fcγ receptors, and thus ablation variants can be usedwhen the constant domain (or Fc domain) in the backbone of theheterodimeric antibody is IgG1. Alternatively, or in addition toablation variants in an IgG1 background, mutations at the glycosylationposition 297 (generally to A or S) can significantly ablate binding toFcγRIIIa, for example. Human IgG2 and IgG4 have naturally reducedbinding to the Fcγ receptors, and thus those backbones can be used withor without the ablation variants.

Combination of Heterodimeric and Fc Variants

As will be appreciated by those in the art, all of the recitedheterodimerization variants (including skew and/or pI variants) can beoptionally and independently combined in any way, as long as they retaintheir “strandedness” or “monomer partition”. In addition, all of thesevariants can be combined into any of the heterodimerization formats.

In the case of pI variants, while embodiments finding particular use areshown in the Figures, other combinations can be generated, following thebasic rule of altering the pI difference between two monomers tofacilitate purification.

In addition, any of the heterodimerization variants, skew and pI, arealso independently and optionally combined with Fc ablation variants, Fcvariants, FcRn variants, as generally outlined herein.

H. Useful Formats of the Invention

As will be appreciated by those in the art and discussed more fullybelow, the heterodimeric fusion proteins of the present invention cantake on a wide variety of configurations, as are generally depicted inFIG. 1. Some figures depict “single ended” configurations, where thereis one type of specificity on one “arm” of the molecule and a differentspecificity on the other “arm”. Other figures depict “dual ended”configurations, where there is at least one type of specificity at the“top” of the molecule and one or more different specificities at the“bottom” of the molecule. Thus, the present invention is directed tonovel immunoglobulin compositions that co-engage a different first and asecond antigen.

As will be appreciated by those in the art, the heterodimeric formats ofthe invention can have different valencies as well as be bispecific.That is, heterodimeric antibodies of the invention can be bivalent andbispecific, wherein one target tumor antigen (e.g. CD3) is bound by onebinding domain and the other target tumor antigen (e.g. SSTR2) is boundby a second binding domain. The heterodimeric antibodies can also betrivalent and bispecific, wherein the first antigen is bound by twobinding domains and the second antigen by a second binding domain. As isoutlined herein, when CD3 is one of the target antigens, it ispreferable that the CD3 is bound only monovalently, to reduce potentialside effects.

The present invention utilizes anti-CD3 antigen binding domains incombination with anti-SSTR2 binding domains. As will be appreciated bythose in the art, any collection of anti-CD3 CDRs, anti-CD3 variablelight and variable heavy domains, Fabs and scFvs as depicted in any ofthe Figures (see particularly FIGS. 2 through 7, and FIG. 18) can beused. Similarly, any of the anti-SSTR2 antigen binding domains can beused, whether CDRs, variable light and variable heavy domains, Fabs andscFvs as depicted in any of the Figures (e.g., FIGS. 8 and 10) can beused, optionally and independently combined in any combination.

Bottle Opener

One heterodimeric scaffold that finds particular use in the presentinvention is the “triple F” or “bottle opener” scaffold format as shownin FIG. 1A. In this embodiment, one heavy chain of the antibody containsan single chain Fv (“scFv”, as defined below) and the other heavy chainis a “regular” FAb format, comprising a variable heavy chain and a lightchain. This structure is sometimes referred to herein as “triple F”format (scFv-FAb-Fc) or the “bottle-opener” format, due to a roughvisual similarity to a bottle-opener. The two chains are broughttogether by the use of amino acid variants in the constant regions(e.g., the Fc domain, the CH1 domain and/or the hinge region) thatpromote the formation of heterodimeric antibodies as is described morefully below.

There are several distinct advantages to the present “triple F” format.As is known in the art, antibody analogs relying on two scFv constructsoften have stability and aggregation problems, which can be alleviatedin the present invention by the addition of a “regular” heavy and lightchain pairing. In addition, as opposed to formats that rely on two heavychains and two light chains, there is no issue with the incorrectpairing of heavy and light chains (e.g. heavy 1 pairing with light 2,etc.).

Many of the embodiments outlined herein rely in general on the bottleopener format that comprises a first monomer comprising an scFv,comprising a variable heavy and a variable light domain, covalentlyattached using an scFv linker (charged, in many but not all instances),where the scFv is covalently attached to the N-terminus of a first Fcdomain usually through a domain linker (which, as outlined herein caneither be un-charged or charged). The second monomer of the bottleopener format is a heavy chain, and the composition further comprises alight chain.

In general, in many preferred embodiments, the scFv is the domain thatbinds to the CD3, and the Fab forms a SSTR2 binding domain.

In addition, the Fc domains of the invention generally comprise skewvariants (e.g. a set of amino acid substitutions as shown in FIGS. 3 and8, with particularly useful skew variants being selected from the groupconsisting of S364K/E357Q:L368D/K370S; L368D/K370S:S364K;L368E/K370S:S364K; T411T/E360E/Q362E:D401K; L368D/K370S:S364K/E357L andK370S:S364K/E357Q), optionally ablation variants (including those shownin FIG. 5), optionally charged scFv linkers (including those shown inFIG. 7) and the heavy chain comprises pI variants (including those shownin FIG. 4).

In some embodiments, the bottle opener format includes skew variants, pIvariants, and ablation variants. Accordingly, some embodiments includebottle opener formats that comprise: a) a first monomer (the “scFvmonomer”) that comprises a charged scFv linker (with the +H sequence ofFIG. 7 being preferred in some embodiments), the skew variantsS364K/E357Q, the ablation variants E233P/L234V/L235A/G236del/S267K, andan Fv that binds to CD3 as outlined herein; b) a second monomer (the“Fab monomer”) that comprises the skew variants L368D/K370S, the pIvariants N208D/Q295E/N384D/Q418E/N421D, the ablation variantsE233P/L234V/L235A/G236del/S267K, and a variable heavy domain that, withthe variable light domain, makes up an Fv that binds to SSTR2 asoutlined herein; and c) a light chain.

Exemplary variable heavy and light domains of the scFv that binds to CD3are included in FIGS. 12 and 13. Exemplary variable heavy and lightdomains of the Fv that binds to SSTR2 are included in FIG. 11. In anexemplary embodiment, the SSTR2 binding domain is an H1.143_L1.30 SSTR2binding domain and the scFv that binds to CD3 includes the variableheavy and light domain of an H1.30_L1.47 CD3 binding domain. Otherparticularly useful SSTR2 and CD3 sequence combinations are disclosedFIG. 16.

In some embodiments, the bottle opener format includes skew variants, pIvariants, ablation variants and FcRn variants. Accordingly, someembodiments include bottle opener formats that comprise: a) a firstmonomer (the “scFv monomer”) that comprises a charged scFv linker (withthe +H sequence of FIG. 7 being preferred in some embodiments), the skewvariants S364K/E357Q, the ablation variantsE233P/L234V/L235A/G236del/S267K, the FcRn variants M428L/N434S and an Fvthat binds to CD3 as outlined herein; b) a second monomer (the “Fabmonomer”) that comprises the skew variants L368D/K370S, the pI variantsN208D/Q295E/N384D/Q418E/N421D, the ablation variantsE233P/L234V/L235A/G236del/S267K, the FcRn variants M428L/N434S, and avariable heavy domain that, with the variable light domain, makes up anFv that binds to SSTR2 as outlined herein; and c) a light chain.

Exemplary variable heavy and light domains of scFvs that bind to CD3 areincluded in FIGS. 12 and 13. Exemplary variable heavy and light domainsof the Fv that binds to SSTR2 are included in FIG. 11. In an exemplaryembodiment, the SSTR2 binding domain includes the variable heavy andvariable light domain of a H1.143_L1.30 SSTR2 binding domain and thescFv that binds to CD3 includes the variable heavy and light domain ofan H1.30_L1.47 CD3 binding domain. Other particularly useful SSTR2 andCD3 sequence combinations are disclosed FIG. 16.

FIG. 9 shows some exemplary bottle opener “backbone” sequences that aremissing the Fv sequences that can be used in the present invention. Insome embodiments, any of the vh and vl sequences depicted herein(including all vh and vl sequences depicted in the Figures and SequenceListings, including those directed to SSTR2) can be added to the bottleopener backbone formats of FIG. 9 as the “Fab side”, using any of theanti-CD3 scFv sequences shown in the Figures and Sequence Listings.

For bottle opener backbone 1 from FIG. 9, (optionally including the428L/434S variants), CD binding domain sequences finding particular usein these embodiments include, but are not limited to, CD3 binding domainanti-CD3 H1.30_L1.47, anti-CD3 H1.32_L1.47, anti-CD3 H1.89_L1.47,anti-CD3 H1.90_L1.47, anti-CD3 H1.33_L1.47 and anti-CD3 H1.31_L1.47, aswell as those depicted in FIGS. 12 and 13, attached as the scFv side ofthe backbones shown in FIG. 9.

For bottle opener backbone 1 from FIG. 9, (optionally including the428L/434S variants), SSTR2 binding domain sequences that are ofparticular use in these embodiments include, but are not limited to,anti-SSTR2 H1.143 L1.30; anti-SSTR2 H1 L1.1; anti-SSTR2 H1.107_L1.30;anti-SSTR2 H1.107_L1.67; anti-SSTR2 H1.107 L1.108; anti-SSTR2 H1.107L1.111; anti-SSTR2 H1.107 L1.114; anti-SSTR2 H1.107 L1.102; anti-SSTR2H1.107 L1.110; anti-SSTR2 H1.125 L1.30; anti-SSTR2 H1.125_L1.67;Anti-SSTR2 H1.125_L1.108; anti-SSTR2 H1.125_L1.111; anti-SSTR2 H1.125L1.114; anti-SSTR2 H1.125 L1.102; and anti-SSTR2 H1.125 L1.10.

Particularly useful SSTR2 and CD3 sequence combinations for use withbottle opener backbone 1 from FIG. 9, (optionally including the428L/434S variants), are disclosed in FIG. 16.

In one exemplary embodiment, the bottle opener antibody includes bottleopener “backbone” 1 from FIG. 9, the SSTR2 binding domain includes thevariable heavy and light domain of an H1.143_L1.30 SSTR2 binding domainand the scFv that binds to CD3 includes the variable heavy and lightdomain of an H1.30_L1.47 CD3 binding domain.

mAb-Fv

One heterodimeric scaffold that finds particular use in the presentinvention is the mAb-Fv format shown in FIG. 1H. In this embodiment, theformat relies on the use of a C-terminal attachment of an “extra”variable heavy domain to one monomer and the C-terminal attachment of an“extra” variable light domain to the other monomer, thus forming a thirdantigen binding domain, wherein the Fab portions of the two monomersbind a SSTR2 and the “extra” scFv domain binds CD3.

In this embodiment, the first monomer comprises a first heavy chain,comprising a first variable heavy domain and a first constant heavydomain comprising a first Fc domain, with a first variable light domaincovalently attached to the C-terminus of the first Fc domain using adomain linker (vh1-CH1-hinge-CH2-CH3-[optional linker]-vl2). The secondmonomer comprises a second variable heavy domain of the second constantheavy domain comprising a second Fc domain, and a third variable heavydomain covalently attached to the C-terminus of the second Fc domainusing a domain linker (vj1-CH1-hinge-CH2-CH3-[optional linker]-vh2. Thetwo C-terminally attached variable domains make up a Fv that binds CD3(as it is less preferred to have bivalent CD3 binding). This embodimentfurther utilizes a common light chain comprising a variable light domainand a constant light domain that associates with the heavy chains toform two identical Fabs that bind a SSTR2. As for many of theembodiments herein, these constructs include skew variants, pI variants,ablation variants, additional Fc variants, etc. as desired and describedherein.

The present invention provides mAb-Fv formats where the CD bindingdomain sequences are as shown in FIGS. 12 and 13 and the SequenceListing. The present invention provides mAb-Fv formats wherein the SSTR2binding domain sequences are as shown in FIG. 11 and the SequenceListing. Particularly useful SSTR2 and CD3 sequence combinations for usewith the mAb-Fv format are disclosed FIG. 16.

In addition, the Fc domains of the mAb-Fv format comprise skew variants(e.g. a set of amino acid substitutions as shown in FIGS. 3 and 8, withparticularly useful skew variants being selected from the groupconsisting of S364K/E357Q:L368D/K370S; L368D/K370S:S364K;L368E/K370S:S364K; T411T/E360E/Q362E:D401K; L368D/K370S:S364K/E357L,K370S:S364K/E357Q, T366S/L368A/Y407V:T366W andT366S/L368A/Y407V/Y349C:T366W/S354C), optionally ablation variants(including those shown in FIG. 5), optionally charged scFv linkers(including those shown in FIG. 7) and the heavy chain comprises pIvariants (including those shown in FIG. 4).

In some embodiments, the mAb-Fv format includes skew variants, pIvariants, and ablation variants. Accordingly, some embodiments includemAb-Fv formats that comprise: a) a first monomer that comprises the skewvariants S364K/E357Q, the ablation variantsE233P/L234V/L235A/G236del/S267K, and a first variable heavy domain that,with the first variable light domain of the light chain, makes up an Fvthat binds to SSTR2, and a second variable heavy domain; b) a secondmonomer that comprises the skew variants L368D/K370S, the pI variantsN208D/Q295E/N384D/Q418E/N421D, the ablation variantsE233P/L234V/L235A/G236del/S267K, and a first variable heavy domain that,with the first variable light domain, makes up the Fv that binds toSSTR2 as outlined herein, and a second variable light chain, thattogether with the second variable heavy domain forms an Fv (ABD) thatbinds to CD3; and c) a light chain comprising a first variable lightdomain and a constant light domain.

In some embodiments, the mAb-Fv format includes skew variants, pIvariants, ablation variants and FcRn variants. Accordingly, someembodiments include mAb-Fv formats that comprise: a) a first monomerthat comprises the skew variants S364K/E357Q, the ablation variantsE233P/L234V/L235A/G236del/S267K, the FcRn variants M428L/N434S and afirst variable heavy domain that, with the first variable light domainof the light chain, makes up an Fv that binds to SSTR2, and a secondvariable heavy domain; b) a second monomer that comprises the skewvariants L368D/K370S, the pI variants N208D/Q295E/N384D/Q418E/N421D, theablation variants E233P/L234V/L235A/G236del/S267K, the FcRn variantsM428L/N434S and a first variable heavy domain that, with the firstvariable light domain, makes up the Fv that binds to SSTR2 as outlinedherein, and a second variable light chain, that together with the secondvariable heavy domain of the first monomer forms an Fv (ABD) that bindsCD3; and c) a light chain comprising a first variable light domain and aconstant light domain.

For mAb-Fv sequences that are similar to the mAb-Fv backbone 1 from FIG.10, (optionally including the M428L/434S variants), CD3 binding domainsequences finding particular use in these embodiments include, but arenot limited to, anti-CD3 H1.30_L1.47, anti-CD3 H1.32 L1.47, anti-CD3H1.89 L1.47, anti-CD3 H1.90 L1.47, anti-CD3 H1.33_L1.47 and anti-CD3H1.31_L1.47, as well as those depicted in FIGS. 12 and 13.

For mAb-Fv sequences that are similar to the mAb-Fv backbone 1 from FIG.10, (optionally including the M428L/434S variants), SSTR2 binding domainsequences that are of particular use in these embodiments include, butare not limited to, anti-SSTR2 H1.143 L1.30; anti-SSTR2 H1 L1.1;anti-SSTR2 H1.107 L1.30; anti-SSTR2 H1.107_L1.67; anti-SSTR2H1.107_L1.108; anti-SSTR2 H1.107_L1.11; anti-SSTR2 H1.107 L1.114;anti-SSTR2 H1.107 L1.102; anti-SSTR2 H1.107 L1.110; anti-SSTR2H1.125_L1.30; anti-SSTR2 H1.125_L1.67; Anti-SSTR2 H1.125_L1.108;anti-SSTR2 H1.125 L1.111; anti-SSTR2 H1.125 L1.114; anti-SSTR2 H1.125L1.102; and anti-SSTR2 H1.125_L1.10, as well as those listed in FIGS. 11and 15 and SEQ ID NOs: 68 to 659.

Particularly useful SSTR2 and CD3 sequence combinations for use withmAb-Fv sequences that are similar to the mAb-Fv backbone 1 from FIG. 10,(optionally including the 428L/434S variants), are disclosed FIG. 16.

mAb-scFv

One heterodimeric scaffold that finds particular use in the presentinvention is the mAb-scFv format shown in FIG. 1. In this embodiment,the format relies on the use of a C-terminal attachment of a scFv to oneof the monomers, thus forming a third antigen binding domain, whereinthe Fab portions of the two monomers bind SSTR2 and the “extra” scFvdomain binds CD3. Thus, the first monomer comprises a first heavy chain(comprising a variable heavy domain and a constant domain), with aC-terminally covalently attached scFv comprising a scFv variable lightdomain, an scFv linker and a scFv variable heavy domain in eitherorientation (vh1-CH1-hinge-CH2-CH3-[optional linker]-vh2-scFv linker-vl2or vh1-CH1-hinge-CH2-CH3-[optional linker]-vl2-scFv linker-vh2). Thisembodiment further utilizes a common light chain comprising a variablelight domain and a constant light domain, that associates with the heavychains to form two identical Fabs that bind SSTR2. As for many of theembodiments herein, these constructs include skew variants, pI variants,ablation variants, additional Fc variants, etc. as desired and describedherein.

The present invention provides mAb-scFv formats where the CD bindingdomain sequences are as shown in FIGS. 12 and 13 and the SequenceListing. The present invention provides mAb-scFv formats wherein theSSTR2 binding domain sequences are as shown in FIG. 11 and the SequenceListing. Particularly useful SSTR2 and CD3 sequence combinations for usewith the mAb-scFv format are disclosed FIG. 16.

In addition, the Fc domains of the mAb-scFv format comprise skewvariants (e.g. a set of amino acid substitutions as shown in FIGS. 3 and8, with particularly useful skew variants being selected from the groupconsisting of S364K/E357Q:L368D/K370S; L368D/K370S:S364K;L368E/K370S:S364K; T411T/E360E/Q362E:D401K; L368D/K370S:S364K/E357L,K370S:S364K/E357Q, T366S/L368A/Y407V:T366W andT366S/L368A/Y407V/Y349C:T366W/S354C), optionally ablation variants(including those shown in FIG. 5), optionally charged scFv linkers(including those shown in FIG. 7) and the heavy chain comprises pIvariants (including those shown in FIG. 4).

In some embodiments, the mAb-scFv format includes skew variants, pIvariants, and ablation variants. Accordingly, some embodiments includemAb-scFv formats that comprise: a) a first monomer that comprises theskew variants S364K/E357Q, the ablation variantsE233P/L234V/L235A/G236del/S267K, and a variable heavy domain that, withthe variable light domain of the common light chain, makes up an Fv thatbinds to SSTR2 as outlined herein, and a scFv domain that binds to CD3;b) a second monomer that comprises the skew variants L368D/K370S, the pIvariants N208D/Q295E/N384D/Q418E/N421D, the ablation variantsE233P/L234V/L235A/G236del/S267K, and a variable heavy domain that, withthe variable light domain of the common light chain, makes up an Fv thatbinds to SSTR2 as outlined herein; and c) a common light chaincomprising a variable light domain and a constant light domain.

In some embodiments, the mAb-scFv format includes skew variants, pIvariants, ablation variants and FcRn variants. Accordingly, someembodiments include mAb-scFv formats that comprise: a) a first monomerthat comprises the skew variants S364K/E357Q, the ablation variantsE233P/L234V/L235A/G236del/S267K, the FcRn variants M428L/N434S and avariable heavy domain that, with the variable light domain of the commonlight chain, makes up an Fv that binds to SSTR2 as outlined herein, anda scFv domain that binds to CD3; b) a second monomer that comprises theskew variants L368D/K370S, the pI variantsN208D/Q295E/N384D/Q418E/N421D, the ablation variantsE233P/L234V/L235A/G236del/S267K, the FcRn variants M428L/N434S and avariable heavy domain that, with the variable light domain of the commonlight chain, makes up an Fv that binds to SSTR2 as outlined herein; andc) a common light chain comprising a variable light domain and aconstant light domain.

In mAb-scFv backbone 1 (optionally including M428L/N434S) from FIG. 10,(optionally including the 428L/434S variantsCD3 binding domain sequencesfinding particular use in these embodiments include, but are not limitedto, anti-CD3 H1.30_L1.47, anti-CD3 H1.32 L1.47, anti-CD3 H1.89 L1.47,anti-CD3 H1.90 L1.47, anti-CD3 H1.33_L1.47 and anti-CD3 H1.31_L1.47, aswell as those depicted in FIGS. 12 and 13.

In mAb-scFv backbone 1 (optionally including M428L/N434S) from FIG. 10,(optionally including the 428L/434S variants), SSTR2 binding domainsequences that are of particular use in these embodiments include, butare not limited to, anti-SSTR2 H1.143_L1.30; anti-SSTR2 H1 L1.1;anti-SSTR2 H1.107_L1.30; anti-SSTR2 H1.107 L1.67; anti-SSTR2 H1.107L1.108; anti-SSTR2 H1.107 L1.111; anti-SSTR2 H1.107 L1.114; anti-SSTR2H1.107 L1.102; anti-SSTR2 H1.107 L1.110; anti-SSTR2 H1.125_L1.30;anti-SSTR2 H1.125_L1.67; Anti-SSTR2 H1.125_L1.108; anti-SSTR2 H1.125L1.111; anti-SSTR2 H1.125 L1.114; anti-SSTR2 H1.125 L1.102; andanti-SSTR2 H1.125_L1.10, as well as those listed in FIGS. 11 and 15 andSEQ ID NOs: 68 to 659.

Central-scFv

One heterodimeric scaffold that finds particular use in the presentinvention is the Central-scFv format shown in FIG. 1. In thisembodiment, the format relies on the use of an inserted scFv domain thusforming a third antigen binding domain, wherein the Fab portions of thetwo monomers bind SSTR2 and the “extra” scFv domain binds CD3. The scFvdomain is inserted between the Fc domain and the CH1-Fv region of one ofthe monomers, thus providing a third antigen binding domain.

In this embodiment, one monomer comprises a first heavy chain comprisinga first variable heavy domain, a CH1 domain (and optional hinge) and Fcdomain, with a scFv comprising a scFv variable light domain, an scFvlinker and a scFv variable heavy domain. The scFv is covalently attachedbetween the C-terminus of the CH1 domain of the heavy constant domainand the N-terminus of the first Fc domain using optional domain linkers(vh1-CH1-[optional linker]-vh2-scFv linker-vl2-[optional linkerincluding the hinge]-CH2-CH3, or the opposite orientation for the scFv,vh1-CH1-[optional linker]-vl2-scFv linker-vh2-[optional linker includingthe hinge]-CH2-CH3). The other monomer is a standard Fab side. Thisembodiment further utilizes a common light chain comprising a variablelight domain and a constant light domain, that associates with the heavychains to form two identical Fabs that bind SSTR2. As for many of theembodiments herein, these constructs include skew variants, pI variants,ablation variants, additional Fc variants, etc. as desired and describedherein.

The present invention provides central-scFv formats where the CD3binding domain sequences are as shown in FIGS. 12 and 13 and theSequence Listing. The present invention provides central-scFv formatswherein the anti-SSTR2 sequences are as shown in FIG. 11 and theSequence Listing. Particularly useful SSTR2 and CD3 sequencecombinations for use with the central-scFv format are disclosed FIG. 16.

In addition, the Fc domains of the central scFv format comprise skewvariants (e.g. a set of amino acid substitutions as shown in FIGS. 3 and8, with particularly useful skew variants being selected from the groupconsisting of S364K/E357Q:L368D/K370S; L368D/K370S:S364K;L368E/K370S:S364K; T411T/E360E/Q362E:D401K; L368D/K370S:S364K/E357L,K370S:S364K/E357Q, T366S/L368A/Y407V:T366W andT366S/L368A/Y407V/Y349C:T366W/S354C), optionally ablation variants(including those shown in FIG. 5), optionally charged scFv linkers(including those shown in FIG. 7) and the heavy chain comprises pIvariants (including those shown in FIG. 4).

In some embodiments, the central-scFv format includes skew variants, pIvariants, and ablation variants. Accordingly, some embodiments includecentral scFv formats that comprise: a) a first monomer that comprisesthe skew variants S364K/E357Q, the ablation variantsE233P/L234V/L235A/G236del/S267K, and a variable heavy domain that, withthe variable light domain of the light chain, makes up an Fv that bindsto SSTR2 as outlined herein, and an scFv domain that binds to CD3; b) asecond monomer that comprises the skew variants L368D/K370S, the pIvariants N208D/Q295E/N384D/Q418E/N421D, the ablation variantsE233P/L234V/L235A/G236del/S267K, and a variable heavy domain that, withvariable light domain of the light chain, makes up an Fv that binds toSSTR2 as outlined herein; and c) a light chain comprising a variablelight domain and a constant light domain.

In some embodiments, the central-scFv format includes skew variants, pIvariants, ablation variants and FcRn variants. Accordingly, someembodiments include central scFv formats that comprise: a) a firstmonomer that comprises the skew variants S364K/E357Q, the ablationvariants E233P/L234V/L235A/G236del/S267K, the FcRn variants M428L/N434Sand a variable heavy domain that, with the variable light domain of thelight chain, makes up an Fv that binds to SSTR2 as outlined herein, andan scFv domain that binds to CD3; b) a second monomer that comprises theskew variants L368D/K370S, the pI variantsN208D/Q295E/N384D/Q418E/N421D, the ablation variantsE233P/L234V/L235A/G236del/S267K, the FcRn variants M428L/N434S and avariable heavy domain that, with variable light domain of the lightchain, makes up an Fv that binds to SSTR2 as outlined herein; and c) alight chain comprising a variable light domain and a constant lightdomain.

For central-scFv sequences that are similar to/utilize the bottle openerbackbone 1 of FIG. 9, (optionally including M428L/N434S), CD3 bindingdomain sequences finding particular use in these embodiments include,but are not limited to, anti-CD3 H1.30_L1.47, anti-CD3 H1.32_L1.47,anti-CD3 H1.89_L1.47, anti-CD3 H1.90_L1.47, anti-CD3 H1.33_L1.47 andanti-CD3 H1.31_L1.47, as well as those depicted in FIGS. 12 and 13.

For central-scFv sequences that are similar to/utilize the bottle openerbackbone 1 of FIG. 9, (optionally including the M428L/434S variants),SSTR2 binding domain sequences that are of particular use in theseembodiments include, but are not limited to, anti-SSTR2 H1.143_L1.30;anti-SSTR2 H1 L1.1; anti-SSTR2 H1.107_L1.30; anti-SSTR2 H1.107 L1.67;anti-SSTR2 H1.107 L1.108; anti-SSTR2 H1.107 L1.111; anti-SSTR2H1.107_L1.114; anti-SSTR2 H1.107_L1.102; anti-SSTR2 H1.107_L1.110;anti-SSTR2 H1.125 L1.30; anti-SSTR2 H1.125 L1.67; Anti-SSTR2 H1.125L1.108; anti-SSTR2 H1.125 L1.111; anti-SSTR2 H1.125 L1.114; anti-SSTR2H1.125 L1.102; and anti-SSTR2 H1.125_L1.10, as well as those listed inFIGS. 11 and 15 and SEQ ID NOs: 68 to 659.

Central-Fv

One heterodimeric scaffold that finds particular use in the presentinvention is the Central-Fv format shown in FIG. 1G. In this embodiment,the format relies on the use of an inserted Fv domain (i.e., the centralFv domain) thus forming a third antigen binding domain, wherein the Fabportions of the two monomers bind a SSTR2 and the “central Fv” domainbinds CD3. The scFv domain is inserted between the Fc domain and theCH1-Fv region of the monomers, thus providing a third antigen bindingdomain, wherein each monomer contains a component of the scFv (e.g. onemonomer comprises a variable heavy domain and the other a variable lightdomain).

In this embodiment, one monomer comprises a first heavy chain comprisinga first variable heavy domain, a CH1 domain, and Fc domain and anadditional variable light domain. The light domain is covalentlyattached between the C-terminus of the CH1 domain of the heavy constantdomain and the N-terminus of the first Fc domain using domain linkers(vh1-CH1-[optional linker]-vl2-hinge-CH2-CH3). The other monomercomprises a first heavy chain comprising a first variable heavy domain,a CH1 domain and Fc domain and an additional variable heavy domain(vh1-CH1-[optional linker]-vh2-hinge-CH2-CH3). The light domain iscovalently attached between the C-terminus of the CH1 domain of theheavy constant domain and the N-terminus of the first Fc domain usingdomain linkers.

This embodiment further utilizes a common light chain comprising avariable light domain and a constant light domain, that associates withthe heavy chains to form two identical Fabs that bind a SSTR2. As formany of the embodiments herein, these constructs include skew variants,pI variants, ablation variants, additional Fc variants, etc. as desiredand described herein.

The present invention provides central-Fv formats where the CD3 bindingdomain sequences are as shown in FIGS. 12 and 13 and the SequenceListing. The present invention provides central-Fv formats wherein theSSTR2 binding domain sequences are as shown in FIG. 11 and the SequenceListing. Particularly useful SSTR2 and CD3 sequence combinations for usewith the central-Fv format are disclosed FIG. 16.

For central-Fv formats, CD3 binding domain sequences finding particularuse in these embodiments include, but are not limited to, anti-CD3 H1.30L1.47, anti-CD3 H1.32 L1.47, anti-CD3 H1.89 L1.47, anti-CD3 H1.90 L1.47,anti-CD3 H1.33 L1.47 and anti-CD3 H1.31_L1.47, as well as those depictedin FIGS. 12 and 13.

For central-Fv formats, SSTR2 binding domain sequences that are ofparticular use in these embodiments include, but are not limited to,anti-SSTR2 H1.143 L1.30; anti-SSTR2 H1 L1.1; anti-SSTR2 H1.107 L1.30;anti-SSTR2 H1.107_L1.67; anti-SSTR2 H1.107_L1.108; anti-SSTR2H1.107_L1.11; anti-SSTR2 H1.107 L1.114; anti-SSTR2 H1.107 L1.102;anti-SSTR2 H1.107 L1.110; anti-SSTR2 H1.125 L1.30; anti-SSTR2 H1.125L1.67; Anti-SSTR2 H1.125 L1.108; anti-SSTR2 H1.125 L1.111; anti-SSTR2H1.125 L1.114; anti-SSTR2 H1.125 L1.102; and anti-SSTR2 H1.125_L1.10, aswell as those listed in FIGS. 11 and 15 and SEQ ID NOs: 68 to 659.

One Armed Central-scFv

One heterodimeric scaffold that finds particular use in the presentinvention is the one armed central-scFv format shown in FIG. 1. In thisembodiment, one monomer comprises just an Fc domain, while the othermonomer uses an inserted scFv domain thus forming the second antigenbinding domain. In this format, either the Fab portion binds a SSTR2 andthe scFv binds CD3 or vice versa. The scFv domain is inserted betweenthe Fc domain and the CH1-Fv region of one of the monomers.

In this embodiment, one monomer comprises a first heavy chain comprisinga first variable heavy domain, a CH1 domain and Fc domain, with a scFvcomprising a scFv variable light domain, an scFv linker and a scFvvariable heavy domain. The scFv is covalently attached between theC-terminus of the CH1 domain of the heavy constant domain and theN-terminus of the first Fc domain using domain linkers. The secondmonomer comprises an Fc domain. This embodiment further utilizes a lightchain comprising a variable light domain and a constant light domain,that associates with the heavy chain to form a Fab. As for many of theembodiments herein, these constructs include skew variants, pI variants,ablation variants, additional Fc variants, etc. as desired and describedherein.

The present invention provides central-Fv formats where the CD3 bindingdomain sequences are as shown in FIGS. 12 and 13 and the SequenceListing. The present invention provides central-Fv formats wherein theSSTR2 binding domain sequences are as shown in FIG. 11 and the SequenceListing. Particularly useful SSTR2 and CD3 sequence combinations for usewith the central-Fv format are disclosed FIG. 16.

In addition, the Fc domains of the one armed central-scFv formatgenerally include skew variants (e.g. a set of amino acid substitutionsas shown in FIGS. 3 and 8, with particularly useful skew variants beingselected from the group consisting of S364K/E357Q:L368D/K370S;L368D/K370S:S364K; L368E/K370S:S364K; T411T/E360E/Q362E:D401K;L368D/K370S:S364K/E357L, K370S:S364K/E357Q, T366S/L368A/Y407V:T366W andT366S/L368A/Y407V/Y349C:T366W/S354C), optionally ablation variants(including those shown in FIG. 5), optionally charged scFv linkers(including those shown in FIG. 7) and the heavy chain comprises pIvariants (including those shown in FIG. 4).

In some embodiments, the one armed central-scFv format includes skewvariants, pI variants, and ablation variants. Accordingly, someembodiments of the one armed central-scFv formats comprise: a) a firstmonomer that comprises the skew variants S364K/E357Q, the ablationvariants E233P/L234V/L235A/G236del/S267K, and a variable heavy domainthat, with the variable light domain of the light chain, makes up an Fvthat binds to SSTR2 as outlined herein, and a scFv domain that binds toCD3; b) a second monomer that includes an Fc domain having the skewvariants L368D/K370S, the pI variants N208D/Q295E/N384D/Q418E/N421D, theablation variants E233P/L234V/L235A/G236del/S267K; and c) a light chaincomprising a variable light domain and a constant light domain.

In some embodiments, the one armed central-scFv format includes skewvariants, pI variants, ablation variants and FcRn variants. Accordingly,some embodiments of the one armed central-scFv formats comprise: a) afirst monomer that comprises the skew variants S364K/E357Q, the ablationvariants E233P/L234V/L235A/G236del/S267K, the FcRn variants M428L/N434Sand a variable heavy domain that, with the variable light domain of thelight chain, makes up an Fv that binds to SSTR2 as outlined herein, anda scFv domain that binds to CD3; b) a second monomer that includes an Fcdomain having the skew variants L368D/K370S, the pI variantsN208D/Q295E/N384D/Q418E/N421D, the ablation variantsE233P/L234V/L235A/G236del/S267K, and the FcRn variants M428L/N434S; andc) a light chain comprising a variable light domain and a constant lightdomain.

For one armed central-scFv formats, CD3 binding domain sequences findingparticular use include, but are not limited to, anti-CD3 H1.30_L1.47,anti-CD3 H1.32_L1.47, anti-CD3 H1.89 L1.47, anti-CD3 H1.90 L1.47,anti-CD3 H1.33 L1.47 and anti-CD3 H1.31_L1.47, as well as those depictedin FIGS. 12 and 13.

For one armed central-scFv formats, SSTR2 binding domain sequences thatare of particular use include, but are not limited to, anti-SSTR2H1.143_L1.30; anti-SSTR2 H1 L1.1; anti-SSTR2 H1.107 L1.30; anti-SSTR2H1.107 L1.67; anti-SSTR2 H1.107 L1.108; anti-SSTR2 H1.107 L1.111;anti-SSTR2 H1.107 L1.114; anti-SSTR2 H1.107 L1.102; anti-SSTR2 H1.107L1.110; anti-SSTR2 H1.125 L1.30; anti-SSTR2 H1.125 L1.67; Anti-SSTR2H1.125 L1.108; anti-SSTR2 H1.125 L1.111; anti-SSTR2 H1.125_L1.114;anti-SSTR2 H1.125_L1.102; and anti-SSTR2 H1.125_L1.10, as well as thoselisted in FIGS. 11 and 15 and SEQ ID NOs: 68 to 659.

One Armed scFv-mAb

One heterodimeric scaffold that finds particular use in the presentinvention is the one armed scFv-mAb format shown in FIG. 1D. In thisembodiment, one monomer comprises just an Fc domain, while the othermonomer uses a scFv domain attached at the N-terminus of the heavychain, generally through the use of a linker: vh-scFvlinker-vl-[optional domain linker]-CH1-hinge-CH2-CH3 or (in the oppositeorientation) vl-scFv linker-vh-[optional domainlinker]-CH1-hinge-CH2-CH3. In this format, the Fab portions each bindSSTR2 and the scFv binds CD3. This embodiment further utilizes a lightchain comprising a variable light domain and a constant light domain,that associates with the heavy chain to form a Fab. As for many of theembodiments herein, these constructs include skew variants, pI variants,ablation variants, additional Fc variants, etc. as desired and describedherein.

The present invention provides one armed scFv-mAb formats where the CD3binding domain sequences are as shown in FIGS. 12 and 13 and theSequence Listing. The present invention provides one armed scFv-mAbformats wherein the SSTR2 binding domain sequences are as shown in FIG.11 and the Sequence Listing. Particularly useful SSTR2 and CD3 sequencecombinations for use with the one armed scFv-mAb format are disclosedFIG. 16.

In addition, the Fc domains of the one armed scFv-mAb format generallyinclude skew variants (e.g. a set of amino acid substitutions as shownin FIGS. 3 and 8, with particularly useful skew variants being selectedfrom the group consisting of S364K/E357Q: L368D/K370S;L368D/K370S:S364K; L368E/K370S:S364K; T411T/E360E/Q362E:D401K;L368D/K370S:S364K/E357L, K370S:S364K/E357Q, T366S/L368A/Y407V:T366W andT366S/L368A/Y407V/Y349C:T366W/S354C), optionally ablation variants(including those shown in FIG. 5), optionally charged scFv linkers(including those shown in FIG. 7) and the heavy chain comprises pIvariants (including those shown in FIG. 4).

In some embodiments, the one armed scFv-mAb format includes skewvariants, pI variants, and ablation variants. Accordingly, someembodiments of the one armed scFv-mAb formats comprise: a) a firstmonomer that comprises the skew variants S364K/E357Q, the ablationvariants E233P/L234V/L235A/G236del/S267K, and a variable heavy domainthat, with the variable light domain of the light chain, makes up an Fvthat binds to SSTR2 as outlined herein, and a scFv domain that binds toCD3; b) a second monomer that includes an Fc domain having the skewvariants L368D/K370S, the pI variants N208D/Q295E/N384D/Q418E/N421D, theablation variants E233P/L234V/L235A/G236del/S267K; and c) a light chaincomprising a variable light domain and a constant light domain.

In some embodiments, the one armed scFv-mAb format includes skewvariants, pI variants, ablation variants and FcRn variants. Accordingly,some embodiments one armed scFv-mAb formats comprise: a) a first monomerthat comprises the skew variants S364K/E357Q, the ablation variantsE233P/L234V/L235A/G236del/S267K, the FcRn variants M428L/N434S and avariable heavy domain that, with the variable light domain of the lightchain, makes up an Fv that binds to SSTR2 as outlined herein, and a scFvdomain that binds to CD3; b) a second monomer that includes an Fc domainhaving the skew variants L368D/K370S, the pI variantsN208D/Q295E/N384D/Q418E/N421D, the ablation variantsE233P/L234V/L235A/G236del/S267K, and the FcRn variants M428L/N434S; andc) a light chain comprising a variable light domain and a constant lightdomain.

For one armed scFv-mAb formats, CD3 binding domain sequences findingparticular use include, but are not limited to, anti-CD3 H1.30_L1.47,anti-CD3 H1.32_L1.47, anti-CD3 H1.89_L1.47, anti-CD3 H1.90_L1.47,anti-CD3 H1.33 L1.47 and anti-CD3 H1.31_L1.47, as well as those depictedin FIGS. 12 and 13.

For one armed scFv-mAb formats, SSTR2 binding domain sequences that areof particular use include, but are not limited to, anti-SSTR2H1.143_L1.30; anti-SSTR2 H1 L1.1; anti-SSTR2 H1.107_L1.30; anti-SSTR2H1.107_L1.67; anti-SSTR2 H1.107 L1.108; anti-SSTR2 H1.107 L1.111;anti-SSTR2 H1.107 L1.114; anti-SSTR2 H1.107_L1.102; anti-SSTR2H1.107_L1.110; anti-SSTR2 H1.125_L1.30; anti-SSTR2 H1.125 L1.67;Anti-SSTR2 H1.125 L1.108; anti-SSTR2 H1.125 L1.111; anti-SSTR2H1.125_L1.114; anti-SSTR2 H1.125_L1.102; and anti-SSTR2 H1.125_L1.10, aswell as those listed in FIGS. 11 and 15 and SEQ ID NOs: 68 to 659.

scFv-mAb

One heterodimeric scaffold that finds particular use in the presentinvention is the mAb-scFv format shown in FIG. 1E. In this embodiment,the format relies on the use of a N-terminal attachment of a scFv to oneof the monomers, thus forming a third antigen binding domain, whereinthe Fab portions of the two monomers bind SSTR2 and the “extra” scFvdomain binds CD3.

In this embodiment, the first monomer comprises a first heavy chain(comprising a variable heavy domain and a constant domain), with aN-terminally covalently attached scFv comprising a scFv variable lightdomain, an scFv linker and a scFv variable heavy domain in eitherorientation ((vh1-scFv linker-vl1-[optional domainlinker]—vh2-CH1-hinge-CH2-CH3) or (with the scFv in the oppositeorientation) ((vl1-scFv linker-vh1-[optional domainlinker]-vh2-CH1-hinge-CH2-CH3)). This embodiment further utilizes acommon light chain comprising a variable light domain and a constantlight domain that associates with the heavy chains to form two identicalFabs that bind SSTR2. As for many of the embodiments herein, theseconstructs include skew variants, pI variants, ablation variants,additional Fc variants, etc. as desired and described herein.

The present invention provides scFv-mAb formats where the CD3 bindingdomain sequences are as shown in FIGS. 12 and 13 and the SequenceListing. The present invention provides scFv-mAb formats wherein theSSTR2 binding domain sequences are as shown in FIG. 11 and the SequenceListing. Particularly useful SSTR2 and CD3 sequence combinations for usewith the scFv-mAb format are disclosed FIG. 16.

In addition, the Fc domains of the scFv-mAb format generally includeskew variants (e.g. a set of amino acid substitutions as shown in FIGS.3 and 8, with particularly useful skew variants being selected from thegroup consisting of S364K/E357Q:L368D/K370S; L368D/K370S:S364K;L368E/K370S:S364K; T411T/E360E/Q362E: D401K; L368D/K370S:S364K/E357L,K370S:S364K/E357Q, T366S/L368A/Y407V:T366W andT366S/L368A/Y407V/Y349C:T366W/S354C), optionally ablation variants(including those shown in FIG. 5), optionally charged scFv linkers(including those shown in FIG. 7) and the heavy chain comprises pIvariants (including those shown in FIG. 4).

In some embodiments, the scFv-mAb format includes skew variants, pIvariants, and ablation variants. Accordingly, some embodiments includescFv-mAb formats that comprise: a) a first monomer that comprises theskew variants S364K/E357Q, the ablation variantsE233P/L234V/L235A/G236del/S267K, and a variable heavy domain that, withthe variable light domain of the common light chain, makes up an Fv thatbinds to SSTR2 as outlined herein, and a scFv domain that binds to CD3;b) a second monomer that comprises the skew variants L368D/K370S, the pIvariants N208D/Q295E/N384D/Q418E/N421D, the ablation variantsE233P/L234V/L235A/G236del/S267K, and a variable heavy domain that, withthe variable light domain of the common light chain, makes up an Fv thatbinds to SSTR2 as outlined herein; and c) a common light chaincomprising a variable light domain and a constant light domain.

In some embodiments, the scFv-mAb format includes skew variants, pIvariants, ablation variants and FcRn variants. Accordingly, someembodiments include scFv-mAb formats that comprise: a) a first monomerthat comprises the skew variants S364K/E357Q, the ablation variantsE233P/L234V/L235A/G236del/S267K, the FcRn variants M428L/N434S and avariable heavy domain that, with the variable light domain of the commonlight chain, makes up an Fv that binds to SSTR2 as outlined herein, anda scFv domain that binds to CD3; b) a second monomer that comprises theskew variants L368D/K370S, the pI variantsN208D/Q295E/N384D/Q418E/N421D, the ablation variantsE233P/L234V/L235A/G236del/S267K, the FcRn variants M428L/N434S and avariable heavy domain that, with the variable light domain of the commonlight chain, makes up an Fv that binds to SSTR2 as outlined herein; andc) a common light chain comprising a variable light domain and aconstant light domain.

For the mAb-scFv format backbone 1 (optionally including M428L/N434S)from FIG. 10, CD3 binding domain sequences finding particular useinclude, but are not limited to, anti-CD3 H1.30 L1.47, anti-CD3 H1.32L1.47, anti-CD3 H1.89 L1.47, anti-CD3 H1.90_L1.47, anti-CD3 H1.33 L1.47and anti-CD3 H1.31_L1.47, as well as those depicted in FIGS. 12 and 13

For the mAb-scFv format backbone 1 (optionally including M428L/N434S)from FIG. 10, SSTR2 binding domain sequences that are of particular useinclude, but are not limited to, anti-SSTR2 H1.143 L1.30; anti-SSTR2 H1L1.1; anti-SSTR2 H1.107 L1.30; anti-SSTR2 H1.107_L1.67; anti-SSTR2H1.107_L1.108; anti-SSTR2 H1.107L1.111; anti-SSTR2 H1.107_L1.114;anti-SSTR2 H1.107_L1.102; anti-SSTR2 H1.107_L1.110; anti-SSTR2 H1.125L1.30; anti-SSTR2 H1.125 L1.67; Anti-SSTR2 H1.125 L1.108; anti-SSTR2H1.125L1.111; anti-SSTR2 H1.125_L1.114; anti-SSTR2 H1.125_L1.102; andanti-SSTR2 H1.125_L1.10, as well as those listed in FIGS. 11 and 15 andSEQ ID NOs: 68 to 659.

Dual scFv Formats

The present invention also provides dual scFv formats as are known inthe art and shown in FIG. 1B. In this embodiment, the SSTR2×CD3heterodimeric bispecific antibody is made up of two scFv-Fc monomers(both in either (vh-scFv linker-vl-[optional domain linker]-CH2-CH3)format or (vl-scFv linker-vh-[optional domain linker]-CH2-CH3) format,or with one monomer in one orientation and the other in the otherorientation.

The present invention provides dual scFv formats where the CD3 bindingdomain sequences are as shown in FIGS. 12 and 13 and the SequenceListing. The present invention provides dual scFv formats wherein theSSTR2 binding domain sequences are as shown in FIG. 11 and the SequenceListing. Particularly useful SSTR2 and CD3 sequence combinations for usewith the dual scFv format are disclosed FIG. 16.

In some embodiments, the dual scFv format includes skew variants, pIvariants, and ablation variants. Accordingly, some embodiments includedual scFv formats that comprise: a) a first monomer that comprises theskew variants S364K/E357Q, the ablation variantsE233P/L234V/L235A/G236del/S267K, and a first scFv that binds either CD3or SSTR2; and b) a second monomer that comprises the skew variantsL368D/K370S, the pI variants N208D/Q295E/N384D/Q418E/N421D, the ablationvariants E233P/L234V/L235A/G236del/S267K, and a second scFv that bindseither CD3 or SSTR2.

In some embodiments, the dual scFv format includes skew variants, pIvariants, ablation variants and FcRn variants. In some embodiments, thedual scFv format includes skew variants, pI variants, and ablationvariants. Accordingly, some embodiments include dual scFv formats thatcomprise: a) a first monomer that comprises the skew variantsS364K/E357Q, the ablation variants E233P/L234V/L235A/G236del/S267K, theFcRn variants M428L/N434S and a first scFv that binds either CD3 orSSTR2; and b) a second monomer that comprises the skew variantsL368D/K370S, the pI variants N208D/Q295E/N384D/Q418E/N421D, the ablationvariants E233P/L234V/L235A/G236del/S267K, the FcRn variants M428L/N434Sand a second scFv that binds either CD3 or SSTR2.

For the dual scFv format, CD3 binding domain sequences findingparticular use include, but are not limited to, anti-CD3 H1.30 L1.47,anti-CD3 H1.32_L1.47, anti-CD3 H1.89 L1.47, anti-CD3 H1.90 L1.47,anti-CD3 H1.33 L1.47 and anti-CD3 H1.31 L1.47, as well as those depictedin FIGS. 12 and 13.

For the dual scFv format, SSTR2 binding domain sequences that are ofparticular use include, but are not limited to, anti-SSTR2 H1.143_L1.30;anti-SSTR2 H1 L1.1; anti-SSTR2 H1.107 L1.30; anti-SSTR2 H1.107 L1.67;anti-SSTR2 H1.107 L1.108; anti-SSTR2 H1.107 L1.111; anti-SSTR2 H1.107L1.114; anti-SSTR2 H1.107 L1.102; anti-SSTR2 H1.107 L1.110; anti-SSTR2H1.125 L1.30; anti-SSTR2 H1.125_L1.67; Anti-SSTR2 H1.125_L1.108;anti-SSTR2 H1.125_L1.111; anti-SSTR2 H1.125 L1.114; anti-SSTR2 H1.125L1.102; and anti-SSTR2 H1.125 L1.10, as well as those listed in FIGS. 11and 15 and SEQ ID NOs: 68 to 659.

Monospecific, Monoclonal Antibodies

As will be appreciated by those in the art, the novel Fv sequencesoutlined herein can also be used in both monospecific antibodies (e.g.“traditional monoclonal antibodies”) or non-heterodimeric bispecificformats. Accordingly, the present invention provides monoclonal(monospecific) antibodies comprising the 6 CDRs and/or the vh and vlsequences from the figures, generally with IgG1, IgG2, IgG3 or IgG4constant regions, with IgG1, IgG2 and IgG4 (including IgG4 constantregions comprising a S228P amino acid substitution) finding particularuse in some embodiments. That is, any sequence herein with a “H_L”designation can be linked to the constant region of a human IgG1antibody.

I. Antigen Binding Domains to Target Antigens

The bispecific antibodies of the invention have two different antigenbinding domains (ABDs) that bind to two different target checkpointantigens (“target pairs”), in either bivalent, bispecific formats ortrivalent, bispecific formats as generally shown in FIG. 1. Note thatgenerally these bispecific antibodies are named “anti-SSTR2×anti-CD3”,or generally simplistically or for ease (and thus interchangeably) as“SSTR2×CD3”, etc. for each pair. Note that unless specified herein, theorder of the antigen list in the name does not confer structure; that isa SSTR2×CD3 bottle opener antibody can have the scFv bind to SSTR2 orCD3, although in some cases, the order specifies structure as indicated.

As is more fully outlined herein, these combinations of ABDs can be in avariety of formats, as outlined below, generally in combinations whereone ABD is in a Fab format and the other is in an scFv format. Asdiscussed herein and shown in FIG. 1, some formats use a single Fab anda single scFv (FIGS. 1A, C and D), and some formats use two Fabs and asingle scFv (FIGS. 1E, F, and I).

Antigen Binding Domains

As discussed herein, the subject heterodimeric antibodies include twoantigen binding domains (ABDs), each of which bind to SSTR2 or CD3. Asoutlined herein, these heterodimeric antibodies can be bispecific andbivalent (each antigen is bound by a single ABD, for example, in theformat depicted in FIG. 1A), or bispecific and trivalent (one antigen isbound by a single ABD and the other is bound by two ABDs, for example asdepicted in FIG. 1F).

In addition, in general, one of the ABDs comprises a scFv as outlinedherein, in an orientation from N- to C-terminus of vh-scFv linker-vl orvl-scFv linker-vh. One or both of the other ABDs, according to theformat, generally is a Fab, comprising a vh domain on one protein chain(generally as a component of a heavy chain) and a vl on another proteinchain (generally as a component of a light chain).

The invention provides a number of ABDs that bind to a number ofdifferent checkpoint proteins, as outlined below. As will be appreciatedby those in the art, any set of 6 CDRs or vh and vl domains can be inthe scFv format or in the Fab format, which is then added to the heavyand light constant domains, where the heavy constant domains comprisevariants (including within the CH1 domain as well as the Fc domain). ThescFv sequences contained in the sequence listing utilize a particularcharged linker, but as outlined herein, uncharged or other chargedlinkers can be used, including those depicted in FIG. 7.

In addition, as discussed above, the numbering used in the SequenceListing for the identification of the CDRs is Kabat, however, differentnumbering can be used, which will change the amino acid sequences of theCDRs as shown in Table 1.

For all of the variable heavy and light domains listed herein, furthervariants can be made. As outlined herein, in some embodiments the set of6 CDRs can have from 0, 1, 2, 3, 4 or 5 amino acid modifications (withamino acid substitutions finding particular use), as well as changes inthe framework regions of the variable heavy and light domains, as longas the frameworks (excluding the CDRs) retain at least about 80, 85 or90% identity to a human germline sequence selected from those listed inFIG. 1 of U.S. Pat. No. 7,657,380, which Figure and Legend isincorporated by reference in its entirety herein. Thus, for example, theidentical CDRs as described herein can be combined with differentframework sequences from human germline sequences, as long as theframework regions retain at least 80, 85 or 90% identity to a humangermline sequence selected from those listed in FIG. 1 of U.S. Pat. No.7,657,380. Alternatively, the CDRs can have amino acid modifications(e.g. from 1, 2, 3, 4 or 5 amino acid modifications in the set of CDRs(that is, the CDRs can be modified as long as the total number ofchanges in the set of 6 CDRs is less than 6 amino acid modifications,with any combination of CDRs being changed; e.g. there may be one changein vlCDR1, two in vhCDR2, none in vhCDR3, etc.)), as well as havingframework region changes, as long as the framework regions retain atleast 80, 85 or 90% identity to a human germline sequence selected fromthose listed in FIG. 1 of U.S. Pat. No. 7,657,380.

SSTR2 Antigen Binding Domains

In some embodiments, one of the ABDs binds SSTR2. Suitable sets of 6CDRs and/or vh and vl domains, as well as scFv sequences, are depictedin FIG. 11 and the Sequence Listing. SSTR2 binding domain sequences thatare of particular use include, but are not limited to, anti-SSTR2H1.143_L1.30; anti-SSTR2 H1 L1.1; anti-SSTR2 H1.107 L1.30; anti-SSTR2H1.107 L1.67; anti-SSTR2 H1.107 L1.108; anti-SSTR2 H1.107 L1.111;anti-SSTR2 H1.107 L1.114; anti-SSTR2 H1.107 L1.102; anti-SSTR2H1.107_L1.110; anti-SSTR2 H1.125_L1.30; anti-SSTR2 H1.125_L1.67;Anti-SSTR2 H1.125 L1.108; anti-SSTR2 H1.125 L1.111; anti-SSTR2 H1.125L1.114; anti-SSTR2 H1.125_L1.102; and anti-SSTR2 H1.125_L1.10, as wellas those listed in FIGS. 11 and 15 and SEQ ID NOs: 68 to 659.

As will be appreciated by those in the art, suitable SSTR2 bindingdomains can comprise a set of 6 CDRs as depicted in the Sequence Listingand Figures, either as they are underlined or, in the case where adifferent numbering scheme is used as described herein and as shown inTable 1, as the CDRs that are identified using other alignments withinthe vh and vl sequences of those depicted in FIG. 11. Suitable ABDs canalso include the entire vh and vl sequences as depicted in thesesequences and Figures, used as scFvs or as Fabs. In many of theembodiments herein that contain an Fv to SSTR2, it is the Fab monomerthat binds SSTR2.

In addition to the parental CDR sets disclosed in the figures andsequence listing that form an ABD to SSTR2, the invention providesvariant CDR sets. In one embodiment, a set of 6 CDRs can have 1, 2, 3, 4or 5 amino acid changes from the parental CDRs, as long as the SSTR2 ABDis still able to bind to the target antigen, as measured by at least oneof a Biacore, surface plasmon resonance (SPR) and/or BLI (biolayerinterferometry, e.g. Octet assay) assay, with the latter findingparticular use in many embodiments.

In addition to the parental variable heavy and variable light domainsdisclosed herein that form an ABD to SSTR2, the invention providesvariant vh and vl domains. In one embodiment, the variant vh and vldomains each can have from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acidchanges from the parental vh and vl domain, as long as the ABD is stillable to bind to the target antigen, as measured at least one of aBiacore, surface plasmon resonance (SPR) and/or BLI (biolayerinterferometry, e.g. Octet assay) assay, with the latter findingparticular use in many embodiments. In another embodiment, the variantvh and vl are at least 90, 95, 97, 98 or 99% identical to the respectiveparental vh or vl, as long as the ABD is still able to bind to thetarget antigen, as measured by at least one of a Biacore, surfaceplasmon resonance (SPR) and/or BLI (biolayer interferometry, e.g. Octetassay) assay, with the latter finding particular use in manyembodiments.

Specific preferred embodiments include the H1.143_L1.30 SSTR2 antigenbinding domain, as a “Fab”, included within any of the bottle openerformat backbones of FIG. 9.

CD3 Antigen Binding Domains

In some embodiments, one of the ABDs binds CD3. Suitable sets of 6 CDRsand/or vh and vl domains, as well as scFv sequences, are depicted inFIGS. 12 and 13 and the Sequence Listing. CD3 binding domain sequencesthat are of particular use include, but are not limited to, anti-CD3H1.30_L1.47, anti-CD3 H1.32_L1.47, anti-CD3 H1.89_L1.47, anti-CD3H1.90_L1.47, anti-CD3 H1.33_L1.47 and anti-CD3 H1.31_L1.47, as well asthose depicted in FIGS. 12 and 13.

As will be appreciated by those in the art, suitable CD3 binding domainscan comprise a set of 6 CDRs as depicted in the Sequence Listing andFigures, either as they are underlined or, in the case where a differentnumbering scheme is used as described herein and as shown in Table 1, asthe CDRs that are identified using other alignments within the vh and vlsequences of those depicted in FIG. 11. Suitable ABDs can also includethe entire vh and vl sequences as depicted in these sequences andFigures, used as scFvs or as Fabs. In many of the embodiments hereinthat contain an Fv to CD3, it is the scFv monomer that binds CD3.

In addition to the parental CDR sets disclosed in the figures andsequence listing that form an ABD to CD3, the invention provides variantCDR sets. In one embodiment, a set of 6 CDRs can have 1, 2, 3, 4 or 5amino acid changes from the parental CDRs, as long as the CD3 ABD isstill able to bind to the target antigen, as measured by at least one ofa Biacore, surface plasmon resonance (SPR) and/or BLI (biolayerinterferometry, e.g. Octet assay) assay, with the latter findingparticular use in many embodiments.

In addition to the parental variable heavy and variable light domainsdisclosed herein that form an ABD to CD3, the invention provides variantvh and vl domains. In one embodiment, the variant vh and vl domains eachcan have from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid changes fromthe parental vh and vl domain, as long as the ABD is still able to bindto the target antigen, as measured at least one of a Biacore, surfaceplasmon resonance (SPR) and/or BLI (biolayer interferometry, e.g. Octetassay) assay, with the latter finding particular use in manyembodiments. In another embodiment, the variant vh and vl are at least90, 95, 97, 98 or 99% identical to the respective parental vh or vl, aslong as the ABD is still able to bind to the target antigen, as measuredby at least one of a Biacore, surface plasmon resonance (SPR) and/or BLI(biolayer interferometry, e.g. Octet assay) assay, with the latterfinding particular use in many embodiments.

Specific preferred embodiments include the H1.30_L1.47 CD3 antigenbinding domain, as a “Fab”, included within any of the bottle openerformat backbones of FIG. 9.

J. Useful Embodiments

In one embodiment, a particular combination of skew and pI variants thatfinds use in the present invention is T366S/L368A/Y407V:T366W(optionally including a bridging disulfide,T366S/L368A/Y407V/Y349C:T366W/S354C) with one monomer comprisesQ295E/N384D/Q418E/N481D and the other a positively charged scFv linker(when the format includes an scFv domain). As will be appreciated in theart, the “knobs in holes” variants do not change pI, and thus can beused on either monomer.

K. Nucleic Acids of the Invention

The invention further provides nucleic acid compositions encoding theanti-SSTR2 antibodies provided herein, including, but not limited to,anti-SSTR2×anti-CD3 bispecific antibodies and SSTR2 monospecificantibodies.

As will be appreciated by those in the art, the nucleic acidcompositions will depend on the format and scaffold of the heterodimericprotein. Thus, for example, when the format requires three amino acidsequences, such as for the triple F format (e.g. a first amino acidmonomer comprising an Fc domain and a scFv, a second amino acid monomercomprising a heavy chain and a light chain), three nucleic acidsequences can be incorporated into one or more expression vectors forexpression. Similarly, some formats (e.g. dual scFv formats such asdisclosed in FIG. 1) only two nucleic acids are needed; again, they canbe put into one or two expression vectors.

As is known in the art, the nucleic acids encoding the components of theinvention can be incorporated into expression vectors as is known in theart, and depending on the host cells used to produce the heterodimericantibodies of the invention. Generally the nucleic acids are operablylinked to any number of regulatory elements (promoters, origin ofreplication, selectable markers, ribosomal binding sites, inducers,etc.). The expression vectors can be extra-chromosomal or integratingvectors.

The nucleic acids and/or expression vectors of the invention are thentransformed into any number of different types of host cells as is wellknown in the art, including mammalian, bacterial, yeast, insect and/orfungal cells, with mammalian cells (e.g. CHO cells), finding use in manyembodiments.

In some embodiments, nucleic acids encoding each monomer and theoptional nucleic acid encoding a light chain, as applicable depending onthe format, are each contained within a single expression vector,generally under different or the same promoter controls. In embodimentsof particular use in the present invention, each of these two or threenucleic acids are contained on a different expression vector. As shownherein and in 62/025,931, hereby incorporated by reference, differentvector ratios can be used to drive heterodimer formation. That is,surprisingly, while the proteins comprise first monomer:secondmonomer:light chains (in the case of many of the embodiments herein thathave three polypeptides comprising the heterodimeric antibody) in a1:1:2 ratio, these are not the ratios that give the best results.

The heterodimeric antibodies of the invention are made by culturing hostcells comprising the expression vector(s) as is well known in the art.Once produced, traditional antibody purification steps are done,including an ion exchange chromotography step. As discussed herein,having the pIs of the two monomers differ by at least 0.5 can allowseparation by ion exchange chromatography or isoelectric focusing, orother methods sensitive to isoelectric point. That is, the inclusion ofpI substitutions that alter the isoelectric point (pI) of each monomerso that such that each monomer has a different pI and the heterodimeralso has a distinct pI, thus facilitating isoelectric purification ofthe “triple F” heterodimer (e.g., anionic exchange columns, cationicexchange columns). These substitutions also aid in the determination andmonitoring of any contaminating dual scFv-Fc and mAb homodimerspost-purification (e.g., IEF gels, cIEF, and analytical IEX columns).

L. Biological and Biochemical Functionality of the HeterodimericCheckpoint Antibodies

Generally the bispecific SSTR2×CD3 antibodies of the invention areadministered to patients with cancer, and efficacy is assessed, in anumber of ways as described herein. Thus, while standard assays ofefficacy can be run, such as cancer load, size of tumor, evaluation ofpresence or extent of metastasis, etc., immuno-oncology treatments canbe assessed on the basis of immune status evaluations as well. This canbe done in a number of ways, including both in vitro and in vivo assays.For example, evaluation of changes in immune status (e.g. presence ofICOS+CD4+ T cells following ipi treatment) along with “old fashioned”measurements such as tumor burden, size, invasiveness, LN involvement,metastasis, etc. can be done. Thus, any or all of the following can beevaluated: the inhibitory effects of the checkpoints on CD4+ T cellactivation or proliferation, CD8+T (CTL) cell activation orproliferation, CD8+ T cell-mediated cytotoxic activity and/or CTLmediated cell depletion, NK cell activity and NK mediated celldepletion.

In some embodiments, assessment of treatment is done by evaluatingimmune cell proliferation, using for example, CFSE dilution method, Ki67intracellular staining of immune effector cells, and 3H-Thymidineincorporation method,

In some embodiments, assessment of treatment is done by evaluating theincrease in gene expression or increased protein levels ofactivation-associated markers, including one or more of: CD25, CD69,CD137, ICOS, PD1, GITR, OX40, and cell degranulation measured by surfaceexpression of CD107A.

In general, gene expression assays are done as is known in the art.

In general, protein expression measurements are also similarly done asis known in the art.

In some embodiments, assessment of treatment is done by assessingcytotoxic activity measured by target cell viability detection viaestimating numerous cell parameters such as enzyme activity (includingprotease activity), cell membrane permeability, cell adherence, ATPproduction, co-enzyme production, and nucleotide uptake activity.Specific examples of these assays include, but are not limited to,Trypan Blue or PI staining, 51Cr or 35S release method, LDH activity,MTT and/or WST assays, Calcein-AM assay, Luminescent based assay, andothers.

In some embodiments, assessment of treatment is done by assessing T cellactivity measured by cytokine production, measure either intracellularlyin culture supernatant using cytokines including, but not limited to,IFNγ, TNFα, GM-CSF, IL2, IL6, IL4, IL5, IL10, IL13 using well knowntechniques.

Accordingly, assessment of treatment can be done using assays thatevaluate one or more of the following: (i) increases in immune response,(ii) increases in activation of αβ and/or γδ T cells, (iii) increases incytotoxic T cell activity, (iv) increases in NK and/or NKT cellactivity, (v) alleviation of αβ and/or γδ T-cell suppression, (vi)increases in pro-inflammatory cytokine secretion, (vii) increases inIL-2 secretion; (viii) increases in interferon-γ production, (ix)increases in Th1 response, (x) decreases in Th2 response, (xi) decreasesor eliminates cell number and/or activity of at least one of regulatoryT cells (Tregs.

Assays to Measure Efficacy

In some embodiments, T cell activation is assessed using a MixedLymphocyte Reaction (MLR) assay as is known in the art. An increase inactivity indicates immunostimulatory activity. Appropriate increases inactivity are outlined below.

In one embodiment, the signaling pathway assay measures increases ordecreases in immune response as measured for an example byphosphorylation or de-phosphorylation of different factors, or bymeasuring other post translational modifications.

An increase in activity indicates immunostimulatory activity.Appropriate increases in activity are outlined below.

In one embodiment, the signaling pathway assay measures increases ordecreases in activation of αβ and/or γδ T cells as measured for anexample by cytokine secretion or by proliferation or by changes inexpression of activation markers like for an example CD137, CD107a, PD1,etc. An increase in activity indicates immunostimulatory activity.Appropriate increases in activity are outlined below.

In one embodiment, the signaling pathway assay measures increases ordecreases in cytotoxic T cell activity as measured for an example bydirect killing of target cells like for an example cancer cells or bycytokine secretion or by proliferation or by changes in expression ofactivation markers like for an example CD137, CD107a, PD1, etc. Anincrease in activity indicates immunostimulatory activity. Appropriateincreases in activity are outlined below.

In one embodiment, the signaling pathway assay measures increases ordecreases in NK and/or NKT cell activity as measured for an example bydirect killing of target cells like for an example cancer cells or bycytokine secretion or by changes in expression of activation markerslike for an example CD107a, etc. An increase in activity indicatesimmunostimulatory activity. Appropriate increases in activity areoutlined below.

In one embodiment, the signaling pathway assay measures increases ordecreases in αβ and/or γδ T-cell suppression, as measured for an exampleby cytokine secretion or by proliferation or by changes in expression ofactivation markers like for an example CD137, CD107a, PD1, etc. Anincrease in activity indicates immunostimulatory activity. Appropriateincreases in activity are outlined below.

In one embodiment, the signaling pathway assay measures increases ordecreases in pro-inflammatory cytokine secretion as measured for exampleby ELISA or by Luminex or by Multiplex bead based methods or byintracellular staining and FACS analysis or by Alispot etc. An increasein activity indicates immunostimulatory activity. Appropriate increasesin activity are outlined below.

In one embodiment, the signaling pathway assay measures increases ordecreases in IL-2 secretion as measured for example by ELISA or byLuminex or by Multiplex bead based methods or by intracellular stainingand FACS analysis or by Alispot etc. An increase in activity indicatesimmunostimulatory activity. Appropriate increases in activity areoutlined below.

In one embodiment, the signaling pathway assay measures increases ordecreases in interferon-γ production as measured for example by ELISA orby Luminex or by Multiplex bead based methods or by intracellularstaining and FACS analysis or by Alispot etc. An increase in activityindicates immunostimulatory activity. Appropriate increases in activityare outlined below.

In one embodiment, the signaling pathway assay measures increases ordecreases in Th1 response as measured for an example by cytokinesecretion or by changes in expression of activation markers. An increasein activity indicates immunostimulatory activity. Appropriate increasesin activity are outlined below.

In one embodiment, the signaling pathway assay measures increases ordecreases in Th2 response as measured for an example by cytokinesecretion or by changes in expression of activation markers. An increasein activity indicates immunostimulatory activity. Appropriate increasesin activity are outlined below.

In one embodiment, the signaling pathway assay measures increases ordecreases cell number and/or activity of at least one of regulatory Tcells (Tregs), as measured for example by flow cytometry or by IHC. Adecrease in response indicates immunostimulatory activity. Appropriatedecreases are the same as for increases, outlined below.

In one embodiment, the signaling pathway assay measures increases ordecreases in M2 macrophages cell numbers, as measured for example byflow cytometry or by IHC. A decrease in response indicatesimmunostimulatory activity. Appropriate decreases are the same as forincreases, outlined below.

In one embodiment, the signaling pathway assay measures increases ordecreases in M2 macrophage pro-tumorigenic activity, as measured for anexample by cytokine secretion or by changes in expression of activationmarkers. A decrease in response indicates immunostimulatory activity.Appropriate decreases are the same as for increases, outlined below.

In one embodiment, the signaling pathway assay measures increases ordecreases in N2 neutrophils increase, as measured for example by flowcytometry or by IHC. A decrease in response indicates immunostimulatoryactivity. Appropriate decreases are the same as for increases, outlinedbelow.

In one embodiment, the signaling pathway assay measures increases ordecreases in N2 neutrophils pro-tumorigenic activity, as measured for anexample by cytokine secretion or by changes in expression of activationmarkers. A decrease in response indicates immunostimulatory activity.Appropriate decreases are the same as for increases, outlined below.

In one embodiment, the signaling pathway assay measures increases ordecreases in inhibition of T cell activation, as measured for an exampleby cytokine secretion or by proliferation or by changes in expression ofactivation markers like for an example CD137, CD107a, PD1, etc. Anincrease in activity indicates immunostimulatory activity. Appropriateincreases in activity are outlined below.

In one embodiment, the signaling pathway assay measures increases ordecreases in inhibition of CTL activation as measured for an example bydirect killing of target cells like for an example cancer cells or bycytokine secretion or by proliferation or by changes in expression ofactivation markers like for an example CD137, CD107a, PD1, etc. Anincrease in activity indicates immunostimulatory activity. Appropriateincreases in activity are outlined below.

In one embodiment, the signaling pathway assay measures increases ordecreases in αβ and/or γδ T cell exhaustion as measured for an exampleby changes in expression of activation markers. A decrease in responseindicates immunostimulatory activity. Appropriate decreases are the sameas for increases, outlined below.

In one embodiment, the signaling pathway assay measures increases ordecreases αβ and/or γδ T cell response as measured for an example bycytokine secretion or by proliferation or by changes in expression ofactivation markers like for an example CD137, CD107a, PD1, etc. Anincrease in activity indicates immunostimulatory activity. Appropriateincreases in activity are outlined below.

In one embodiment, the signaling pathway assay measures increases ordecreases in stimulation of antigen-specific memory responses asmeasured for an example by cytokine secretion or by proliferation or bychanges in expression of activation markers like for an example CD45RA,CCR7 etc. An increase in activity indicates immunostimulatory activity.Appropriate increases in activity are outlined below.

In one embodiment, the signaling pathway assay measures increases ordecreases in apoptosis or lysis of cancer cells as measured for anexample by cytotoxicity assays such as for an example MTT, Cr release,Calcine AM, or by flow cytometry based assays like for an example CFSEdilution or propidium iodide staining etc. An increase in activityindicates immunostimulatory activity. Appropriate increases in activityare outlined below.

In one embodiment, the signaling pathway assay measures increases ordecreases in stimulation of cytotoxic or cytostatic effect on cancercells. as measured for an example by cytotoxicity assays such as for anexample MTT, Cr release, Calcine AM, or by flow cytometry based assayslike for an example CFSE dilution or propidium iodide staining etc. Anincrease in activity indicates immunostimulatory activity. Appropriateincreases in activity are outlined below.

In one embodiment, the signaling pathway assay measures increases ordecreases direct killing of cancer cells as measured for an example bycytotoxicity assays such as for an example MTT, Cr release, Calcine AM,or by flow cytometry based assays like for an example CFSE dilution orpropidium iodide staining etc. An increase in activity indicatesimmunostimulatory activity. Appropriate increases in activity areoutlined below.

In one embodiment, the signaling pathway assay measures increases ordecreases Th17 activity as measured for an example by cytokine secretionor by proliferation or by changes in expression of activation markers.An increase in activity indicates immunostimulatory activity.Appropriate increases in activity are outlined below.

In one embodiment, the signaling pathway assay measures increases ordecreases in induction of complement dependent cytotoxicity and/orantibody dependent cell-mediated cytotoxicity, as measured for anexample by cytotoxicity assays such as for an example MTT, Cr release,Calcine AM, or by flow cytometry based assays like for an example CFSEdilution or propidium iodide staining etc. An increase in activityindicates immunostimulatory activity. Appropriate increases in activityare outlined below.

In one embodiment, T cell activation is measured for an example bydirect killing of target cells like for an example cancer cells or bycytokine secretion or by proliferation or by changes in expression ofactivation markers like for an example CD137, CD107a, PD1, etc. ForT-cells, increases in proliferation, cell surface markers of activation(e.g. CD25, CD69, CD137, PD1), cytotoxicity (ability to kill targetcells), and cytokine production (e.g. IL-2, IL-4, IL-6, IFNγ, TNF-α,IL-10, IL-17A) would be indicative of immune modulation that would beconsistent with enhanced killing of cancer cells.

In one embodiment, NK cell activation is measured for example by directkilling of target cells like for an example cancer cells or by cytokinesecretion or by changes in expression of activation markers like for anexample CD107a, etc. For NK cells, increases in proliferation,cytotoxicity (ability to kill target cells and increases CD107a,granzyme, and perforin expression), cytokine production (e.g. IFNγ andTNF), and cell surface receptor expression (e.g. CD25) would beindicative of immune modulation that would be consistent with enhancedkilling of cancer cells.

In one embodiment, γδ T cell activation is measured for example bycytokine secretion or by proliferation or by changes in expression ofactivation markers.

In one embodiment, Th1 cell activation is measured for example bycytokine secretion or by changes in expression of activation markers.

Appropriate increases in activity or response (or decreases, asappropriate as outlined above), are increases of 10%, 20%, 30%, 40%,50%, 60%, 70%, 80%, 90%, 95% or 98 to 99% percent over the signal ineither a reference sample or in control samples, for example testsamples that do not contain an antibody of the invention. Similarly,increases of at least one-, two-, three-, four- or five-fold as comparedto reference or control samples show efficacy.

M. Treatments

Once made, the compositions of the invention find use in a number ofapplications. SSTR2 is high expressed in neuroendocrine tumors (NETs,e.g., lung, GI, pancreatic, pituitary, medullary cancers, prostate,pancreatic lungcarcinoids, osteosarcoma, bronchial, thymus) as well asnon-NETs (breast, lung, colarectal, ovarian, cervial cancers).

Accordingly, the heterodimeric compositions of the invention find use inthe treatment of such SSTR2 positive cancers.

Antibody Compositions for In Vivo Administration

Formulations of the antibodies used in accordance with the presentinvention are prepared for storage by mixing an antibody having thedesired degree of purity with optional pharmaceutically acceptablecarriers, excipients or stabilizers (Remington's Pharmaceutical Sciences16th edition, Osol, A. Ed. [1980]), in the form of lyophilizedformulations or aqueous solutions. Acceptable carriers, excipients, orstabilizers are nontoxic to recipients at the dosages and concentrationsemployed, and include buffers such as phosphate, citrate, and otherorganic acids; antioxidants including ascorbic acid and methionine;preservatives (such as octadecyldimethylbenzyl ammonium chloride;hexamethonium chloride; benzalkonium chloride, benzethonium chloride;phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol);low molecular weight (less than about 10 residues) polypeptides;proteins, such as serum albumin, gelatin, or immunoglobulins;hydrophilic polymers such as polyvinylpyrrolidone; amino acids such asglycine, glutamine, asparagine, histidine, arginine, or lysine;monosaccharides, disaccharides, and other carbohydrates includingglucose, mannose, or dextrins; chelating agents such as EDTA; sugarssuch as sucrose, mannitol, trehalose or sorbitol; salt-formingcounter-ions such as sodium; metal complexes (e.g. Zn-proteincomplexes); and/or non-ionic surfactants such as TWEEN™, PLURONICS™ orpolyethylene glycol (PEG).

Administrative Modalities

The antibodies and chemotherapeutic agents of the invention areadministered to a subject, in accord with known methods, such asintravenous administration as a bolus or by continuous infusion over aperiod of time.

Treatment Modalities

In the methods of the invention, therapy is used to provide a positivetherapeutic response with respect to a disease or condition. By“positive therapeutic response” is intended an improvement in thedisease or condition, and/or an improvement in the symptoms associatedwith the disease or condition. For example, a positive therapeuticresponse would refer to one or more of the following improvements in thedisease: (1) a reduction in the number of neoplastic cells; (2) anincrease in neoplastic cell death; (3) inhibition of neoplastic cellsurvival; (5) inhibition (i.e., slowing to some extent, preferablyhalting) of tumor growth; (6) an increased patient survival rate; and(7) some relief from one or more symptoms associated with the disease orcondition.

Positive therapeutic responses in any given disease or condition can bedetermined by standardized response criteria specific to that disease orcondition. Tumor response can be assessed for changes in tumormorphology (i.e., overall tumor burden, tumor size, and the like) usingscreening techniques such as magnetic resonance imaging (MRI) scan,x-radiographic imaging, computed tomographic (CT) scan, bone scanimaging, endoscopy, and tumor biopsy sampling including bone marrowaspiration (BMA) and counting of tumor cells in the circulation.

In addition to these positive therapeutic responses, the subjectundergoing therapy may experience the beneficial effect of animprovement in the symptoms associated with the disease.

Treatment according to the present invention includes a “therapeuticallyeffective amount” of the medicaments used. A “therapeutically effectiveamount” refers to an amount effective, at dosages and for periods oftime necessary, to achieve a desired therapeutic result.

A therapeutically effective amount may vary according to factors such asthe disease state, age, sex, and weight of the individual, and theability of the medicaments to elicit a desired response in theindividual. A therapeutically effective amount is also one in which anytoxic or detrimental effects of the antibody or antibody portion areoutweighed by the therapeutically beneficial effects.

A “therapeutically effective amount” for tumor therapy may also bemeasured by its ability to stabilize the progression of disease. Theability of a compound to inhibit cancer may be evaluated in an animalmodel system predictive of efficacy in human tumors.

Alternatively, this property of a composition may be evaluated byexamining the ability of the compound to inhibit cell growth or toinduce apoptosis by in vitro assays known to the skilled practitioner. Atherapeutically effective amount of a therapeutic compound may decreasetumor size, or otherwise ameliorate symptoms in a subject. One ofordinary skill in the art would be able to determine such amounts basedon such factors as the subject's size, the severity of the subject'ssymptoms, and the particular composition or route of administrationselected.

Dosage regimens are adjusted to provide the optimum desired response(e.g., a therapeutic response). For example, a single bolus may beadministered, several divided doses may be administered over time or thedose may be proportionally reduced or increased as indicated by theexigencies of the therapeutic situation. Parenteral compositions may beformulated in dosage unit form for ease of administration and uniformityof dosage. Dosage unit form as used herein refers to physically discreteunits suited as unitary dosages for the subjects to be treated; eachunit contains a predetermined quantity of active compound calculated toproduce the desired therapeutic effect in association with the requiredpharmaceutical carrier.

The specification for the dosage unit forms of the present invention aredictated by and directly dependent on (a) the unique characteristics ofthe active compound and the particular therapeutic effect to beachieved, and (b) the limitations inherent in the art of compoundingsuch an active compound for the treatment of sensitivity in individuals.

The efficient dosages and the dosage regimens for the bispecificantibodies used in the present invention depend on the disease orcondition to be treated and may be determined by the persons skilled inthe art.

An exemplary, non-limiting range for a therapeutically effective amountof an bispecific antibody used in the present invention is about 0.1-100mg/kg.

All cited references are herein expressly incorporated by reference intheir entirety.

Whereas particular embodiments of the invention have been describedabove for purposes of illustration, it will be appreciated by thoseskilled in the art that numerous variations of the details may be madewithout departing from the invention as described in the appendedclaims.

Examples

Examples are provided below to illustrate the present invention. Theseexamples are not meant to constrain the present invention to anyparticular application or theory of operation. For all constant regionpositions discussed in the present invention, numbering is according tothe EU index as in Kabat (Kabat et al., 1991, Sequences of Proteins ofImmunological Interest, 5th Ed., United States Public Health Service,National Institutes of Health, Bethesda, entirely incorporated byreference). Those skilled in the art of antibodies will appreciate thatthis convention consists of nonsequential numbering in specific regionsof an immunoglobulin sequence, enabling a normalized reference toconserved positions in immunoglobulin families. Accordingly, thepositions of any given immunoglobulin as defined by the EU index willnot necessarily correspond to its sequential sequence.

General and specific scientific techniques are outlined in USPublications 2015/0307629, 2014/0288275 and WO2014/145806, all of whichare expressly incorporated by reference in their entirety andparticularly for the techniques outlined therein.

Example 1: Generation of Anti-SSTR2×Anti-CD3 Bispecific Antibodies 1A:Generation of Anti-SSTR2 Fab Arm

The parental variable region of an anti-SSTR2 antibody was engineeredfor use as a component of anti-STTR2×anti-CD3 bispecific antibodies ofthe invention. Humanization of murine VH and VL regions was performed aspreviously described in U.S. Pat. No. 7,657,380, issued Feb. 2, 2010.Amino acid substitutions were made via QuikChange (Stratagene, CedarCreek, Tx.) mutagenesis to attempt to identify variants with improvedproperties.

1B: Bispecifics Antibody Production

Cartoon schematics of anti-SSTR2×anti-CD3 bispecific formats are shownin FIG. 1. Exemplary antibodies were generated with anti-SSTR2 Fab armsderived from anti-SSTR2 antibodies engineered as described above andanti-CD3 scFv arms. Exemplary anti-SSTR2×anti-CD3 bottle openerantibodies XENP018087 and XENP018907 are shown in FIGS. 14 and 15,respectively. DNA encoding the three chains needed for bispecificexpression were either generated by gene synthesis (Blue HeronBiotechnology, Bothell, Wash.) and standard subcloning into theexpression vector pTT5 techniques or by QuikChange mutagenesis. DNA wastransfected into HEK293E cells for expression, and the resultingproteins were purified from the supernatant using protein A affinity (GEHealthcare) and cation exchange chromatography. Cation exchangechromatography purification was performed using a HiTrap SP HP column(GE Healthcare) with a wash/equilibration buffer of 50 mM MES, pH 6.0and an elution buffer of 50 mM MES, pH 6.0+1 M NaCl linear gradient.

1C: Anti-SSTR2 Antibody Bispecific Binding.

Cell surface binding of anti-SSTR2 antibodies and exemplaryanti-SSTR2×anti-CD3 bispecific antibodies were assessed using humanSSTR2-transfected CHO cells. Cells were incubated with indicated testarticles for 45 minutes on ice and centrifuged. Cells were resuspendedwith staining buffer containing phycoerythrin (PE) labeled secondaryantibody (2 μg/mL; goat anti-human IgG) and then incubated for 45minutes on ice. Cells were centrifuged twice and then resuspsended withstaining buffer. Binding was measured by flow cytometry (FIGS. 17A-P).

Example 2: Characterization of Exemplary Anti-SSTR2×Anti-CD3 BispecificAntibodies

2A: In Vitro Characterization of Exemplary Anti-SSTR2×Anti-CD3Bispecific Antibodies Exemplary anti-SSTR2×anti-CD3 Fab-scFv-Fcbispecifics were characterized in vitro for redirected T cellcytotoxicity (RTCC) on SSTR2 transfected CHO cells (FIGS. 18A-D) andSSTR2-positive TT cells (a human thyroid medullary carcinoma cell line;FIGS. 19A-C). RTCC was determined by measuring lactate dehydrogenase(LDH) levels. As shown in these figures, anti-SSTR2×anti-CD3 Fab-scFv-Fcbispecifics exhibited a high percentage of RTCC in the SSTR2-transfectedCHO cells, as well as the human cancer cell lines as compared tocontrols.

2B: In Vivo Characterization of Exemplary Anti-SSTR2×Anti-CD3 BispecificAntibodies

In a first study, cynomolgus monkeys (n=3) were administered two (at 0and 3 weeks) intravenous (i.v.) doses of either 0.03 mg/kg XENP18087 or1 mg/kg XENP18088. The effects of these anti-SSTR2×anti-CD3 bispecificantibodies on CD4⁺ and CD8⁺ T cell activation as indicated by CD69expression (FIG. 20A) and CD4⁺ and CD8⁺ T cell distribution (FIG. 20B)were subsequently assessed.

In a second study, cynomolgus monkeys (n=3) were administered a singleintravenous (i.v.) dose of anti-SSTR2×anti-CD3 bispecific antibodies:0.06 mg/kg XENP 18087, 0.1 mg/kg XENP18907, 0.5 mg/kg XENP 18907, or 2mg/kg XENP18907. The effects of these anti-SSTR2×anti-CD3 bispecificantibodies on CD4⁺ and CD8⁺ T cell activation (CD69 upregulation, FIG.21A) and CD4⁺ and CD8⁺ T cell redistribution (cell counts, FIG. 21B)were assessed. In addition, a glucose tolerance test (GTT) was conducted(FIGS. 21C and 21D) to assess the ability of the tested subjects tobreakdown glucose. For the GTT, blood samples were collected at 8different time points: predose, 5, 10, 20, 30, 40, 60, and 90 minutesafter dextrose administration. As shown in these studies, CD4⁺ and CD8⁺were rapidly redistributed from the blood during each treatment withsubsequence recovery and normalization after dosing (FIG. 21B). T cellswere activated immediately upon dosing (FIG. 21A) and then subsequentlysubsided, coincident with T cell redistribution.

In a third study, cynomolgus monkeys (n=3) were administered two (at 0and 1 week) intravenous (i.v.) doses of either 0.001 or 0.01 mg/kgXENP18087. The effects of these anti-SSTR2×anti-CD3 bispecificantibodies on CD4⁺ and CD8⁺ T cell activation (FIGS. 22A-B) and CD4⁺ andCD8⁺ T cell distribution (FIGS. 22C-D) were subsequently assessed usingCD69 expression, a marker of T cell activation. From these monkeys,serum IL-6 and TNF levels were assayed (FIGS. 22 E-F). As shown in thesestudies, CD4⁺ and CD8⁺ were rapidly redistributed from the blood duringeach treatment with subsequent recovery and normalization after dosing.T cells were activated immediately upon each administration in adose-dependent manner and then subsequently subsided, coincident with Tcell redistribution. IL-6 and TNF cytokine release correlated with Tcell activation.

Example 3: Evaluation OF XmAb18087 3A: Specific Binding of XmAb18087 forHuman and Cynomolgus SSTR2

Cell surface binding of XmAb18087 and control anti-RSV×anti-CD3bispecific antibody (XENP13245) were assessed usinghumanSSTR2-transfected CHO cells and cynoSSTR2-transfected CHO cells.Cell surface binding was also assessed using parental CHO cells ascontrol. Binding was measured by flow cytometry using phycoerythrin (PE)labeled secondary antibody as generally described in Example 1C.

XmAb18087 not only bound cell surface human SSTR2 (FIG. 23A) but wasalso cross-reactive with cynomolgus SSTR2 (FIG. 23B), while the controlanti-RSV×anti-CD3 bispecific antibody XENP 13245 did not bind eitherhuman SSTR2 or cynomolgus SSTR2-transfected CHO cells. The data furthershows that XmAb18087 did not bind untransfected parental CHO cells (FIG.23C) demonstrating the specificity of XmAb18087.

3B: Redirected T Cell Cytotoxicity by XmAb18087

XmAb18087 was characterized in vitro for redirected T cell cytotoxicity(RTCC) of SSTR2-transfected CHO cells (FIG. 24A), SSTR2-positive TTcells (a medullary thyroid carcinoma cell line; FIGS. 24B and 25), A549cells (a lung adenocarcinoma cell line; FIGS. 24C and 25) anduntransfected parental CHO cell as a control (FIG. 24A). Ananti-RSV×anti-CD3 bispecific antibody (XENP13245) and bivalentanti-SSTR2 mAb were included as controls (FIG. 24D).

Target cells and human PBMCs were incubated with XmAb18087 or XENP13245for 24 hours at an E:T ratio of 10 or 20:1. RTCC was determined bymeasuring lactate dehydrogenase (LDH) levels.

As shown in these figures, XmAb18087 exhibited a high percentage of RTCCin the SSTR2 transfected CHO cells (FIG. 24A) as well as the humancancer cell lines (FIGS. 28B-C and 24D) as compared to the controlanti-CD3 bispecific antibody XENP13245 and control bivalent anti-SSTR2mAb (FIG. 25). Furthermore, the data show that XmAb18087 did not exhibitRTCC in untransfected parental CHO cells (FIG. 24A).

T cell activation by XmAb18087 was also investigated in the experimentswith SSTR2-transfected CHO cells and TT cells by evaluating the surfaceexpression of CD69 on CD8⁺ and CD4⁺ T cells by flow cytometry (FIG.26A-B). As shown in the figures, XmAb18087 activates CD8⁺ and CD4+ Tcells to a much higher level than the control anti-CD3 bispecificantibody XENP13245. This demonstrates the XmAb18087 eliminates SSTR2⁺target cells by inducing T cell activation.

3C: XmAb18087 Exhibits Anti-Tumor Activity in NSG Mice Engrafted withA549 Lung Carcinoma Cells and Human PBMC

Twenty-five NOD scid gamma (NSG) mice were each engrafted with 1×10⁶A549-RedFLuc tumor cells (0.1 mL volume subcutaneous injection) on Day−7. On Day 0, mice were engrafted intraperitoneally with 10×10⁶ humanPBMCs. After PBMC engraftment on Day 0, XmAb18087 was dosed weekly (Days0, 7, and 14) by intraperitoneal injection at 3.0 mg/kg (control micewere dosed with PBS). Study design is further summarized in FIG. 27.Tumor growth was monitored by measuring total flux per mouse using an invitro imaging system (IVIS® Lumina III).

As shown in FIGS. 28 and 29, treatment with 3 mg/kg XmAb18087substantially suppresses A549 local tumor growth as compared totreatment with PBS.

3D: Characterization of XmAb18087 in Cynomolgus Monkeys

In a further study, cynomolgus monkeys (n=3) were administered a singleintravenous (i.v.) dose of XmAb18087 or control anti-RSV×anti-CD3bispecific antibody (XENP13245). The effects of these bispecificantibodies on CD4⁺ and CD8⁺ T cell activation (CD69 upregulation; FIGS.31A-B), and cytokine (IL-6 and TNF) release (FIGS. 32A-B) were assessed.

As shown in the figures, CD4⁺ and CD8⁺ T cells were rapidlyredistributed from the blood following treatment with XmAb18087 (FIGS.30A and B, as compared to treatment with XENP 13245) with subsequentrecovery and normalizing after dosing. T cells were activatedimmediately upon dosing with XmAb18087 (as compared to dosing withXENP13245) and then subsequently subsided, coincident with T cellredistribution. Further, IL-6 and TNF cytokine release correlated with Tcell activation (FIGS. 32A-B).

3E: Characterization of XmAb18087 in NSG Mice

In another study to investigate dose-response, 60 NSG mice wereengrafted with 1×106 A549-RedFLuc tumor cells (0.1 mL volumesubcutaneous injection) on Day −7. On Day 0, mice were sorted based ontotal flux and engrafted intraperitoneally with 10×106 human PBMCs andadministered Dose #1 of test articles at the indicated concentrations(12 mice for each concentration). Dose #2 and #3 were administered onDay 8 and Day 15. As above, tumor growth was monitored by measuringtotal flux per mouse using an in vitro imaging system two to three timesper week as depicted in FIG. 33. Additionally, tumor volume was measuredby caliper once to twice per week as depicted in Figure X for Day 18 and22 post Dose #1.

What is claimed is:
 1. A heterodimeric antibody comprising: a) a firstheavy chain comprising: i) a first variant Fc domain; and ii) a singlechain Fv region (scFv), wherein said scFv region comprises a firstvariable heavy domain, a first variable light domain and a charged scFvlinker, wherein said charged scFv linker covalently attaches said firstvariable heavy domain and said first variable light domain; b) a secondheavy chain comprising a VH-CH1-hinge-CH2-CH3 monomer, wherein VH is asecond variable heavy domain and CH2-CH3 is a second variant Fc domain;and c) a light chain comprising a second variable light domain and alight constant domain; wherein said second variant Fc domain comprisesamino acid substitutions N208D/Q295E/N384D/Q418E/N421 D, wherein saidfirst and second variant Fc domains each comprise amino acidsubstitutions E233P/L234V/L235A/G236del/S267K, wherein said firstvariant Fc domain comprises amino acid substitutions S364K/E357Q andsaid second variant Fc domain comprises amino acid substitutionsL368D/K370S, wherein said second variable heavy domain comprises SEQ IDNO: 1071 and said second variable light domain comprises SEQ ID NO:1076, wherein numbering is according to the EU index as in Kabat.
 2. Aheterodimeric antibody according to claim 1, wherein said scFv bindsCD3.
 3. A heterodimeric antibody according to claim 1, wherein saidfirst variable heavy domain and said first variable light domain areselected from the sets comprising: SEQ ID NO: 1 and SEQ ID NO: 5; SEQ IDNO: 10 and SEQ ID NO: 14; SEQ ID NO: 19 and SEQ ID NO: 23; SEQ ID NO: 28and SEQ ID NO: 32; SEQ ID NO: 37 and SEQ ID NO: 41; and SEQ ID NO: 46and SEQ ID NO: 50, respectively.
 4. A heterodimeric antibody accordingto claim 3, wherein said first variable heavy domain comprises SEQ IDNO: 1 and said first variable light domain comprises SEQ ID NO:
 5. 5. Aheteodimeric antibody according to claim 1, wherein theCH1-hinge-CH2-CH3 component of the second heavy chain comprises SEQ IDNO: 1108, said first variant Fc domain comprises SEQ ID NO: 1109 andsaid constant light domain comprises SEQ ID NO:
 1110. 6. A heterodimericantibody according to claim 1, wherein said first heavy chain comprisesSEQ ID NO: 1080, said second heavy chain comprises SEQ ID NO: 1070, andsaid light chain comprises SEQ ID NO:
 1075. 7. A nucleic acidcomposition comprising: a) a first nucleic acid encoding said firstheavy chain of claim 1; b) a second nucleic acid encoding said secondheavy chain of claim 1; and c) a third nucleic acid encoding said lightchain of claim
 1. 8. An expression vector composition comprising: a) afirst expression vector comprising said first nucleic acid of claim 7;b) a second expression vector comprising said second nucleic acid ofclaim 7; and c) a third expression vector comprising said third nucleicacid of claim
 7. 9. A host cell comprising said expression vectorcomposition of claim
 8. 10. A method of making a heterodimeric antibodyaccording to claim 1 comprising culturing said host cell of claim 9under conditions wherein said antibody is expressed, and recovering saidantibody.
 11. A method of treating a neuroendocrine cancer in a subjectin need thereof, comprising administering to said subject aheterodimeric antibody according to claim
 1. 12. A compositioncomprising a somatostatin receptor type 2 (SSTR2) binding domain, saidSSTR2 binding domain comprising a variable heavy domain comprising SEQID NO: 958 and a variable light domain comprising SEQ ID NO:
 962. 13. Anucleic acid composition comprising: a) a first nucleic acid encodingsaid variable heavy domain of claim 12; and b) a second nucleic acidencoding said variable light domain of claim
 12. 14. An expressionvector composition comprising: a) a first expression vector comprisingsaid first nucleic acid of claim 13; and b) a second expression vectorcomprising said second nucleic acid of claim
 13. 15. A host cellcomprising said expression vector composition of claim
 14. 16. A methodof making a composition according to claim 12 comprising culturing saidhost cell of claim 15 under conditions wherein said antibody isexpressed, and recovering said antibody.
 17. A method of treating aneuroendocrine cancer in a subject in need thereof, comprisingadministering to said subject a composition according to claim 12.